Influenza hemagglutinin and neuraminidase variants

ABSTRACT

Polypeptides, polynucleotides, methods, compositions, and vaccines comprising influenza hemagglutinin and neuraminidase variants are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/329,123, filed on Dec. 16, 2011, which is a divisional of U.S. patentapplication Ser. No. 12/858,386, filed on Aug. 17, 2010, which is acontinuation of U.S. patent application Ser. No. 12/262,215, filed onOct. 31, 2008, which is a division and claims the benefit of U.S. patentapplication Ser. No. 11/368,246, filed on Mar. 6, 2006, now U.S. Pat.No. 7,459,162, which claims priority to and benefit of U.S. ProvisionalPatent Application Ser. No. 60/659,832, filed on Mar. 8, 2005, thedisclosures of which are incorporated herein in their entirety for allpurposes.

REFERENCE TO A SEQUENCE LISTING

This application refers to a sequence listing, which is provided as anelectronic document on each of two identical compact discs, labeled“Copy 1” and “Copy 2.” Each compact disk contains a 320,100 byte fileentitled “FL2550US2_seqlist.txt,” created Mar. 6, 2006. This electronicsequence listing file is incorporated in its entirety herein.

BACKGROUND OF THE INVENTION

Vaccines against various and evolving strains of influenza are importantfrom a community health standpoint, as well as commercially, since eachyear numerous individuals are infected with different strains and typesof influenza virus. Infants, the elderly, those without adequate healthcare and immuno-compromised persons are at special risk of death fromsuch infections. Compounding the problem of influenza infections is thatnovel influenza strains evolve readily and can spread between variousspecies, thereby necessitating the continuous production of newvaccines.

Numerous vaccines capable of producing a protective immune responsespecific for different influenza viruses/virus strains have beenproduced for over 50 years and include whole virus vaccines, split virusvaccines, surface antigen vaccines and live attenuated virus vaccines.However, while appropriate formulations of any of these vaccine typesare capable of producing a systemic immune response, live attenuatedvirus vaccines have the advantage of also being able to stimulate localmucosal immunity in the respiratory tract. Considerable work in theproduction of influenza viruses, and fragments thereof, for productionof vaccines has been done by the present inventors and co-workers; see,e.g., U.S. Application No. 60/420,708, filed Oct. 23, 2002, U.S.application Ser. No. 10/423,828, filed Apr. 25, 2003, and U.S.Application No. 60/574,117, filed May 24, 2004, all entitled“Multi-Plasmid System for the Production of Influenza Virus.”

Because of the continual emergence (or re-emergence) or differentinfluenza strains, new influenza vaccines are continually desired. Suchvaccines typically are created using antigenic moieties of the newlyemergent virus strains so, therefore, polypeptides and polynucleotidesof novel, newly emergent, or newly re-emergent virus strains (especiallysequences of antigenic genes) are highly desirable. Furthermore, suchsequences within preferred vectors are also quite highly desired.

The present invention provides new and/or newly isolated influenzahemagglutinin and neuraminidase variants, optionally within preferredvectors, that are capable of use in production of numerous types ofvaccines as well as in research, diagnostics, etc. Numerous otherbenefits will become apparent upon review of the following

SUMMARY OF THE INVENTION

In some aspects herein, the invention comprises an isolated orrecombinant polypeptide that is selected from: the polypeptides encodedby any one of the sequences of the sequence listing, e.g., SEQ ID NO:1through SEQ ID NO:48, any one of the polypeptides encoded by thesequence listing, e.g., SEQ ID NO:49 through SEQ ID NO:96; anypolypeptide that is encoded by a polynucleotide sequence whichhybridizes under highly stringent conditions over substantially theentire length of a polynucleotide sequence of the sequence listing; and,a fragment of any of the above wherein the sequence comprises ahemagglutinin or neuraminidase polypeptide, or a fragment of ahemagglutinin or neuraminidase polypeptide. In various embodiments, theisolated or recombinant polypeptides of the invention are substantiallyidentical to about 300 contiguous amino acid residues of any of theabove polypeptides. In yet other embodiments, the invention comprisesisolated or recombinant polypeptides (comprising hemagglutinin orneuraminidase or fragments of hemagglutinin or neuraminidase), thatcomprise an amino acid sequence that is substantially identical over atleast about 350 amino acids; over at least about 400 amino acids; overat least about 450 amino acids; over at least about 500 amino acids;over at least about 502 amino acids; over at least about 550 aminoacids; over at least about 559 amino acids; over at least about 565amino acids; or over at least about 566 amino acids contiguous of any ofthe polypeptides of claim of any of the above polypeptides. In yet otherembodiments, the invention comprises isolated or recombinantpolypeptides (e.g., comprising neuraminidase, hemagglutinin or fragmentsof neuraminidase or hemagglutinin), that comprise an amino acid sequencethat is substantially identical over at least about 350 amino acids;over at least about 400 amino acids; over at least about 436 aminoacids; over at least about 450 amino acids; over at least about 451amino acids; over at least about 465 amino acids; over at least about466 amino acids; over at least about 469 amino acids; or over at leastabout 470 amino acids contiguous of any of the polypeptides of any ofthe above polypeptides. Of course, in some embodiments, the polypeptidesequence (e.g., as listed in the sequence listing herein, e.g., SEQ IDNO:49 through SEQ ID NO:96) comprises less than 565, 559, etc. aminoacids. In such embodiments, the shorter listed polypeptides optionallycomprise less than 565, 559, etc. amino acids. In yet other embodiments,the polypeptides of the invention optionally comprise fusion proteins,proteins with a leader sequence, a precursor polypeptide, proteins witha secretion signal or a localization signal, or proteins with an epitopetag, an E-tag, or a H is epitope tag, etc. In still other embodiments,the invention comprises a polypeptide comprising a sequence having atleast 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, atleast 99%, at least 99.2%, at least 99.4%, at least 99.6%, at least99.8%, or at least 99.9% sequence identity to at least one polypeptidelisted above (e.g., of SEQ ID NO: 49-96). In some embodiments, suchpolypeptides are immunogenic. The HA sequences of the invention cancomprise both those sequences with unmodified and those with modifiedpolybasic cleavage sites.

In other aspects, the invention comprises a composition with one or morepolypeptide listed above, or fragments thereof. The invention alsoincludes polypeptides that are specifically bound by a polyclonalantisera raised against at least 1 antigen that comprises at least oneamino acid sequence described above (e.g., SEQ ID NO: 49-96), or afragment thereof. Such antibodies specific for the polypeptidesdescribed above are also features of the invention. The polypeptides ofthe invention are optionally immunogenic.

The invention also encompasses immunogenic compositions comprising animmunologically effective amount of one or more of any of thepolypeptides described above as well as methods for stimulating theimmune system of an individual to produce a protective immune responseagainst influenza virus by administering to the individual animmunologically effective amount of any of the above polypeptides (e.g.,SEQ ID NO: 49-96) in a physiologically acceptable carrier.

Additionally, the invention has reassortant influenza virus that encodeone or more of the polypeptides above (e.g., SEQ ID NO: 49-96), inaddition to immunogenic compositions comprising an immunologicallyeffective amount of such recombinant influenza virus. Methods forstimulating the immune system of an individual to produce a protectiveimmune response against influenza virus, through administering animmunologically effective amount of such recombinant influenza virus ina physiologically acceptable carrier are also part of the invention.Such virus can optionally comprise a 6:2 reassortant virus with 6 genesencoding regions from one or more donor virus (e.g. A/AA/6/60, B/AnnArbor/1/66, A/Puerto Rico/8/34, which is more commonly known as PR8),B/Leningrad/14/17/55, B/14/5/1, B/USSR/60/69, B/Leningrad/179/86,B/Leningrad/14/55, or B/England/2608/76 and 2 gene encoding regions(typically and preferably encoding HA and NA or fragments thereof)selected from SEQ ID NO:1 through SEQ ID NO:48 or from similar strains,as defined herein, to those having SEQ ID NO:1-48, etc. Immunogeniccompositions comprising such reassortant (recombinant) virus are alsofeatures of the invention.

In other aspects, the invention comprises an isolated or recombinantnucleic acid that is selected from: any one of the polynucleotidesequences of the sequence listing, e.g., SEQ ID NO:1 through SEQ IDNO:48 (or complementary sequences thereof), any one of thepolynucleotide sequences encoding a polypeptide of the sequence listing,e.g., SEQ ID NO:49 through SEQ ID NO:96 (or complementary polynucleotidesequences thereof), a polynucleotide sequence which hybridizes underhighly stringent conditions over substantially the entire length of anyof the above polynucleotide sequences, and a polynucleotide sequencecomprising all or a fragment of any of the above polynucleotidesequences wherein the sequence encodes a hemagglutinin or neuraminidasepolypeptide or one or more HA or NA fragments. Such nucleic acids can beDNA, RNA, cRNA, DNA:RNA hybrids, single stranded nucleic acid, doublestranded nucleic acid, etc. The invention also includes an isolated orrecombinant nucleic acid (e.g., comprising hemagglutinin or fragmentsthereof), that encodes an amino acid sequence which is substantiallyidentical over at least about 300 amino acids of any of the abovenucleic acids, or over at least about 350 amino acids; over at leastabout 400 amino acids; over at least about 450 amino acids; over atleast about 500 amino acids; over at least about 502 amino acids; overat least about 550 amino acids; over at least about 559 amino acids;over at least about 565 amino acids; or over at least about 566 aminoacids of any of the above nucleic acids. In yet other embodiments, theinvention comprises isolated or recombinant nucleic acids (e.g.,comprising neuraminidase or fragments thereof), that encode an aminoacid sequence that is substantially identical over at least about 350amino acids; over at least about 400 amino acids; over at least about436 amino acids; over at least about 450 amino acids; over at leastabout 451 amino acids; over at least about 465 amino acids; over atleast about 466 amino acids; over at least about 469 amino acids; orover at least about 470 amino acids contiguous of any of thepolypeptides above. Again, in situations wherein the amino acid is lessthan, e.g., 566, 565, 559, etc. in length (e.g., see, Sequence Listingin FIG. 1) then it should be understood that the length is optionallyless than 566, 565, 559, etc. The invention also includes any of theabove nucleic acids that comprise a hemagglutinin or neuraminidasepolypeptide, or one or hemagglutinin or neuraminidase fragments. Otheraspects of the invention include isolated or recombinant nucleic acidsthat encode a polypeptide (optionally a hemagglutinin or neuraminidasepolypeptide) whose sequence has at least 95% identity, at least 96%identity, at least 97% identity, at least 98% identity, at least 98.5%identity, at least 99% identity, at least 99.2% identity, at least 99.4%identity, at least 99.6% identity, at least 99.8% identity, or at least99.9% identity to at least one of the above described polynucleotide.The invention also includes isolated or recombinant nucleic acidsencoding a polypeptide of hemagglutinin or neuraminidase produced bymutating or recombining one or more above described polynucleotidesequence. The polynucleotide sequences of the invention can optionallycomprise one or more of, e.g., a leader sequence, a precursor sequence,or an epitope tag sequence or the like, and can optionally encode afusion protein (e.g., with one or more additional nucleic acidsequences). Such nucleic acids of the invention can optionally encodeimmunogenic polypeptides.

In yet other embodiments, the invention comprises a composition ofmatter having two or more above described nucleic acids or fragmentsthereof (e.g., a library comprising at least about 2, 5, 10, 50 or morenucleic acids). Such compositions can optionally be produced by cleavingone or more above described nucleic acid (e.g., mechanically,chemically, enzymatically with a restriction endonuclease/RNAse/DNAse,etc.). Other compositions of the invention include, e.g., compositionsproduced by incubating one or more above described nucleic acid in thepresence of deoxyribonucleotide triphosphates and a thermostable nucleicacid polymerase Immunogenic compositions having an immunologicallyeffective amount of any of the above nucleic acids are also within thecurrent invention.

Also within the invention are reassortant influenza viruses comprisingany of the above nucleic acids. Such reassortant viruses can (andpreferably are) 6:2 reassortant viruses with 6 gene encoding regionsfrom one or more donor virus (e.g., A/AA/6/60, B/AA/1/66 (also sometimesreferred to herein as B/Ann Arbor/1/66), B/Leningrad/14/17/55, B/14/5/1,B/USSR/60/69, B/Leningrad/179/86, B/Leningrad/14/55, orB/England/2608/76 or A/Puerto Rico/8/34) and 2 gene encoding regionsfrom two sequences above (e.g., from SEQ ID NO:1-48, from similarstrains to those encoded in SEQ ID NO:1-48, etc.). Preferably, such tworegions encode hemagglutinin and/or neuraminidase Immunogeniccompositions with immunologically effective amounts of suchreassortant/recombinant influenza virus are also within purview of thecurrent invention.

Vectors comprising one or more nucleic acid from SEQ ID NO:1-48 (again,also from similar strains to those of the sequence identificationnumbers) or fragments thereof are also within the current invention.Such vectors (e.g., expression vectors) can optionally be plasmids,cosmids, phage, viruses, virus fragments, etc. Especially preferredembodiments comprise plasmid vectors useful in plasmid rescue methods toproduce virus (e.g., typically reassortant/recombinant virus for use invaccines). Such plasmid systems are exampled in, e.g., U.S. ApplicationNo. 60/420,708, filed Oct. 23, 2002, U.S. application Ser. No.10/423,828, filed Apr. 25, 2003, and U.S. Application No. 60/574,117,filed May 24, 2004, all entitled “Multi-Plasmid System for theProduction of Influenza Virus”; Hoffmann, E., 2000, PNAS,97(11):6108-6113; U.S. Published Patent Application No. 20020164770 toHoffmann; and U.S. Pat. No. 6,544,785 issued Apr. 8, 2003 to Palese, etal. Cells transduced, transformed, transfected, etc. with such vectorsare also within the current invention.

The invention also encompasses cells comprising at least one abovedescribed nucleic acid, or a cleaved or amplified fragment or productthereof. Such cells can optionally express a polypeptide encoded by suchnucleic acid. Other embodiments of the invention include vectors (e.g.,plasmids, cosmids, phage, viruses, virus fragments, etc.) comprising anyof above described nucleic acid. Such vectors can optionally comprise anexpression vector. Cells transduced by such vectors are also within thecurrent invention.

In some embodiments, the invention encompasses a virus (e.g., aninfluenza virus) comprising one or more above described nucleic acid(e.g., from SEQ ID NO:1-48 or from similar strains to such andoptionally encoding hemagglutinin and/or neuraminidase), or one or morefragments thereof. Typically, such viruses are reassortant/recombinantviruses Immunogenic compositions comprising such virus are also part ofthe current invention. Such viruses can comprises a reassortant virussuch as a 6:2 reassortment virus (which comprises 6 gene encodingregions from one or more donor virus (e.g., a master donor virus or abackbone virus such as A/AA/6/60, B/AA/1/66, A/Puerto Rico/8/34,B/Leningrad/14/17/55, B/14/5/1, B/USSR/60/69, B/Leningrad/179/86,B/Leningrad/14/55, or B/England/2608/76, etc.) and 2 gene encodingregions from one or more above described nucleotide sequence, or one ormore fragment thereof which can optionally comprise hemagglutinin and/orneuraminidase). Other reassortant/recombinant viruses can comprise 7:1reassortments. Reassortment viruses (optionally live viruses) of theinvention can include donor viruses that are one or more of, e.g.,temperature-sensitive (ts), cold-adapted (ca), or attenuated (att). Forexample, reassortment viruses can comprise, e.g., A/Ann Arbor/6/60,B/Ann Arbor/1/66, A/Puerto Rico/8/34, B/Leningrad/14/17/55, B/14/5/1,B/USSR/60/69, B/Leningrad/179/86, B/Leningrad/14/55, orB/England/2608/76, etc. In many embodiments, the produced viruses arelive viruses (e.g., to be used in vaccines, etc.). Other embodimentsinclude dead or inactivated viruses (e.g., also capable of use invaccines, etc.). Cells comprising any of the above viruses are alsoproducts of the invention.

Methods of producing reassortant/recombinant influenza virus throughculturing a host cell harboring an influenza virus in a suitable culturemedium under conditions permitting expression of nucleic acid; and,isolating or recovering the recombinant influenza virus from one or moreof the host cell or the medium are also part of the invention. Thus,introducing a plurality of vectors having an influenza virus genome intoa population of host cells wherein the vectors comprise at least 6internal genome segments of a first influenza strain (again, e.g.,A/AA/6/60, B/AA/1/66, A/PR/8/34, B/Leningrad/14/17/55, B/14/5/1,B/USSR/60/69, B/Leningrad/179/86, B/Leningrad/14/55, orB/England/2608/76, etc.) and at least one (and preferably two) genomesegments are selected from a second influenza strain (e.g., preferablyone or more nucleic acid as described above, e.g., from SEQ ID NO:1-48or from a similar strain to such or optionally comprising ahemagglutinin and/or neuraminidase, etc.). is a feature of theinvention. Preferably, the first strain of virus is cold-adapted and/ortemperature sensitive and/or attenuated. Also preferably, such virusesare suitable for administration as part of an intranasal vaccineformulation. Of course, other embodiments are suitable foradministration as killed or inactivated vaccine formulations,live/attenuated non-nasal vaccine formulations, etc. The vectors in suchmethods can comprise influenza A viruses and/or influenza B viruses.Host cells for such methods can optionally comprise, e.g., Vero cells,PER.C6 cells (deposited under ECACC No. 96022940), MDCK cells, 293Tcells, COS cells, etc. Typical embodiments do not comprise helperviruses in the method and yet other typical embodiments comprise eightplasmid vectors to contain the influenza genome.

In other embodiments herein, the invention comprises immunogeniccompositions having an immunologically effective amount of the abovedescribed recombinant influenza virus (e.g., a live virus). Otherembodiments include methods for stimulating the immune system of anindividual to produce a protective immune response against influenzavirus by administering to the individual an immunologically effectiveamount of the recombinant influenza virus of described above (optionallyin a physiologically effective carrier).

Other aspects of the invention include methods of producing an isolatedor recombinant polypeptide by culturing any host cell above, in asuitable culture medium under conditions permitting expression ofnucleic acid and, isolating the polypeptide from one or more of the hostcell or the medium in which it is grown.

Immunogenic compositions are also features of the invention. Forexample, immunogenic compositions comprising one or more of thepolypeptides and/or nucleic acids described above (e.g., a sequence fromSEQ ID NO:1-96 or from similar strains to such, etc.) and, optionally,an excipient such as a pharmaceutically acceptable excipient or one ormore pharmaceutically acceptable administration component Immunogeniccompositions of the invention can also comprise one or more abovedescribed virus as well (e.g., along with one or more pharmaceuticallyacceptable administration component).

Methods of producing an influenza virus vaccine are also included in theinvention. For example, the invention includes introducing a pluralityof vectors (e.g., plasmid vectors) comprising an influenza genome (e.g.,influenza A or B) into a population of host cells that is capable ofsupporting replication of such virus, culturing the cells, recovering aplurality of influenza viruses and providing one or morepharmaceutically acceptable excipient with such virus to an individual(e.g., one in need of such treatment). Such viruses can optionally becold-adapted and/or temperature sensitive and/or attenuated andpreferably are suitable for administration in an intranasal vaccineformulation. Such methods can include wherein the vectors have at least6 internal genome segments of a first influenza strain and at least onegenome segment (and preferably 2 segments) from another influenza strain(e.g., with sequence selected from SEQ ID NO:1-48 or from similarstrains to such, etc.) which segment optionally codes for an immunogenicinfluenza surface antigen of the second influenza strain.

Methods of producing immunogenic responses in a subject throughadministration of an effective amount of any of the above viruses to asubject are also within the current invention. Additionally, methods ofprophylactic or therapeutic treatment of a viral infection (e.g., viralinfluenza) in a subject through administration of one or more abovedescribed virus in an amount effective to produce an immunogenicresponse against the viral infection are also part of the currentinvention. Subjects for such treatment can include mammals (e.g.,humans). Such methods can also comprise in vivo administration to thesubject as well as in vitro or ex vivo administration to one or morecells of the subject. Additionally, such methods can also compriseadministration of a composition of the virus and a pharmaceuticallyacceptable excipient that is administered to the subject in an amounteffect to prophylactically or therapeutically treat the viral infection.

The invention also comprises compositions of matter having one or moresequence selected from SEQ ID NO:1 through SEQ ID NO:48, and a selectedmaster donor virus, typically wherein the selected sequence and themaster donor virus comprise a 6:2 reassortment, i.e., the HA and NAherein reassorted with the other six influenza genes from the donorvirus. Such donor viruses are typically ca, att, ts influenza strains.For example, typically donor strains can include, e.g., A/AnnArbor/6/60, B/Ann Arbor/1/66, A/Puerto Rico/8/34, B/Leningrad/14/17/55,B/14/5/1, B/USSR/60/69, B/Leningrad/179/86, B/Leningrad/14/55, orB/England/2608/76 and variants thereof. Those of skill in the art willappreciate that typically donor strains can vary from reassortant toreassortant. Thus, those variations are also encompassed within thecurrent invention. Another element of the invention comprises one ormore live attenuated influenza vaccine comprising such compositions,e.g., those having sequences herein reassorted in a 6:2 manner with aselected master donor virus.

Other aspects of the invention include, compositions of mattercomprising a hemagglutinin polynucleotide and/or a neuraminidasepolynucleotide reassorted with one or more master donor virus, againtypically a ca, att, ts influenza virus, wherein the polynucleotidecomprises a same virus strain as one or more virus strain of SEQ ID NO:1through SEQ ID NO:48. Such hemagglutinin and/or neuraminidasepolynucleotide is typically determined to be “within the same strain”when it produces a titer that is within a four-fold range of anothervirus (e.g., ones having the sequences listed herein) as measured by ahemagglutinin inhibition assay. As described below, however, othercommon assays can also be utilized to determine whether polynucleotides(i.e., viruses comprising such) are within the same strain.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying figures and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1AV display sequences of variant hemagglutinin andneuraminidase nucleic acids and polypeptides of the invention.

FIGS. 2A and 2B display an alternative organization of varianthemagglutinin and neuraminidase sequences as found in FIGS. 1A-1AV.

DETAILED DESCRIPTION

The present invention includes polypeptide and polynucleotide sequencesof influenza hemagglutinin and neuraminidase as well as vectors,viruses, vaccines, compositions and the like comprising such sequencesand methods of their use. Additional features of the invention aredescribed in more detail herein.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. The following definitionssupplement those in the art and are directed to the current applicationand are not necessarily to be imputed to any related or unrelated case,e.g., to any commonly owned patent or application. Although any methodsand materials similar or equivalent to those described herein can beused in the practice for testing of the present invention, the preferredmaterials and methods are described herein. Accordingly, the terminologyused herein is for the purpose of describing particular embodimentsonly, and is not intended to be limiting. Additional terms are definedand described throughout.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contextclearly dictates otherwise. Thus, for example, reference to “a virus”includes a plurality of viruses; reference to a “host cell” includesmixtures of host cells, and the like.

The terms “nucleic acid,” “polynucleotide,” “polynucleotide sequence,”and “nucleic acid sequence” refer to single-stranded or double-strandeddeoxyribonucleotide or ribonucleotide polymers, chimeras or analoguesthereof, or a character string representing such, depending on context.As used herein, the term optionally includes polymers of analogs ofnaturally occurring nucleotides having the essential nature of naturalnucleotides in that they hybridize to single-stranded nucleic acids in amanner similar to naturally occurring nucleotides (e.g., peptide nucleicacids). Unless otherwise indicated, a particular nucleic acid sequenceof this invention optionally encompasses complementary sequences inaddition to the sequence explicitly indicated. From any specifiedpolynucleotide sequence, either the given nucleic acid or thecomplementary polynucleotide sequence (e.g., the complementary nucleicacid) can be determined.

The term “nucleic acid” or “polynucleotide” also encompasses anyphysical string of monomer units that can be corresponded to a string ofnucleotides, including a polymer of nucleotides (e.g., a typical DNA orRNA polymer), PNAs, modified oligonucleotides (e.g., oligonucleotidescomprising bases that are not typical to biological RNA or DNA insolution, such as 2′-O-methylated oligonucleotides), and the like. Anucleic acid can be e.g., single-stranded or double-stranded.

A “subsequence” is any portion of an entire sequence, up to andincluding the complete sequence. Typically, a subsequence comprises lessthan the full-length sequence. A “unique subsequence” is a subsequencethat is not found in any previously determined influenza polynucleotideor polypeptide sequence The phrase “substantially identical,” in thecontext of two nucleic acids or polypeptides (e.g., DNAs encoding a HAor NA molecule, or the amino acid sequence of a HA or NA molecule)refers to two or more sequences or subsequences that have at least about90%, preferably 91%, most preferably 92%, 93%, 94%, 95%, 96%, 97%, 98%,98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,99.9% or more nucleotide or amino acid residue identity, when comparedand aligned for maximum correspondence, as measured using a sequencecomparison algorithm or by visual inspection.

The term “variant” with respect to a polypeptide refers to an amino acidsequence that is altered by one or more amino acids with respect to areference sequence. The variant can have “conservative” changes, whereina substituted amino acid has similar structural or chemical properties,e.g., replacement of leucine with isoleucine. Alternatively, a variantcan have “nonconservative” changes, e.g., replacement of a glycine witha tryptophan. Analogous minor variation can also include amino aciddeletion or insertion, or both. Guidance in determining which amino acidresidues can be substituted, inserted, or deleted without eliminatingbiological or immunological activity can be found using computerprograms well known in the art, for example, DNASTAR software. Examplesof conservative substitutions are also described herein.

The term “gene” is used broadly to refer to any nucleic acid associatedwith a biological function. Thus, genes include coding sequences and/orthe regulatory sequences required for their expression. The term “gene”applies to a specific genomic sequence, as well as to a cDNA or an mRNAencoded by that genomic sequence.

The “neuraminidase” polypeptides of the invention show immunologicalcross reactivity with one or more known neuraminidase molecule from aninfluenza virus. The literature is replete with examples of such knownneuraminidases (e.g., in GenBank, in publications from the CDC, etc.).Similarly, the “hemagglutinin” polypeptides of the invention showimmunological cross-reactivity with one or more known hemagglutininmolecule from an influenza virus. Again, the literature is replete withexamples of such known hemagglutinin molecules.

Genes also include non-expressed nucleic acid segments that, forexample, form recognition sequences for other proteins. Non-expressedregulatory sequences include “promoters” and “enhancers,” to whichregulatory proteins such as transcription factors bind, resulting intranscription of adjacent or nearby sequences. A “tissue specific”promoter or enhancer is one that regulates transcription in a specifictissue type or cell type, or types.

“Expression of a gene” or “expression of a nucleic acid” typically meanstranscription of DNA into RNA (optionally including modification of theRNA, e.g., splicing) or transcription of RNA into mRNA, translation ofRNA into a polypeptide (possibly including subsequent modification ofthe polypeptide, e.g., post-translational modification), or bothtranscription and translation, as indicated by the context.

An “open reading frame” or “ORF” is a possible translational readingframe of DNA or RNA (e.g., of a gene), which is capable of beingtranslated into a polypeptide. That is, the reading frame is notinterrupted by stop codons. However, it should be noted that the termORF does not necessarily indicate that the polynucleotide is, in fact,translated into a polypeptide.

The term “vector” refers to the means by which a nucleic acid can bepropagated and/or transferred between organisms, cells, or cellularcomponents. Vectors include plasmids, viruses, bacteriophages,pro-viruses, phagemids, transposons, artificial chromosomes, and thelike, that replicate autonomously or can integrate into a chromosome ofa host cell. A vector can also be a naked RNA polynucleotide, a nakedDNA polynucleotide, a polynucleotide composed of both DNA and RNA withinthe same strand, a poly-lysine-conjugated DNA or RNA, apeptide-conjugated DNA or RNA, a liposome-conjugated DNA, or the like,that is not autonomously replicating. In many, but not all, commonembodiments, the vectors of the present invention are plasmids.

An “expression vector” is a vector, such as a plasmid that is capable ofpromoting expression, as well as replication of a nucleic acidincorporated therein. Typically, the nucleic acid to be expressed is“operably linked” to a promoter and/or enhancer, and is subject totranscription regulatory control by the promoter and/or enhancer.

A “bi-directional expression vector” is characterized by two alternativepromoters oriented in the opposite direction relative to a nucleic acidsituated between the two promoters, such that expression can beinitiated in both orientations resulting in, e.g., transcription of bothplus (+) or sense strand, and negative (−) or antisense strand RNAs.

An “amino acid sequence” is a polymer of amino acid residues (a protein,polypeptide, etc.) or a character string representing an amino acidpolymer, depending on context.

A “polypeptide” is a polymer comprising two or more amino acid residues(e.g., a peptide or a protein). The polymer can optionally comprisemodifications such as glycosylation or the like. The amino acid residuesof the polypeptide can be natural or non-natural and can beunsubstituted, unmodified, substituted or modified.

In the context of the invention, the term “isolated” refers to abiological material, such as a virus, a nucleic acid or a protein, whichis substantially free from components that normally accompany orinteract with it in its naturally occurring environment. The isolatedbiological material optionally comprises additional material not foundwith the biological material in its natural environment, e.g., a cell orwild-type virus. For example, if the material is in its naturalenvironment, such as a cell, the material can have been placed at alocation in the cell (e.g., genome or genetic element) not native tosuch material found in that environment. For example, a naturallyoccurring nucleic acid (e.g., a coding sequence, a promoter, anenhancer, etc.) becomes isolated if it is introduced by non-naturallyoccurring means to a locus of the genome (e.g., a vector, such as aplasmid or virus vector, or amplicon) not native to that nucleic acid.Such nucleic acids are also referred to as “heterologous” nucleic acids.An isolated virus, for example, is in an environment (e.g., a cellculture system, or purified from cell culture) other than the nativeenvironment of wild-type virus (e.g., the nasopharynx of an infectedindividual).

The term “chimeric” or “chimera,” when referring to a virus, indicatesthat the virus includes genetic and/or polypeptide components derivedfrom more than one parental viral strain or source. Similarly, the term“chimeric” or “chimera,” when referring to a viral protein, indicatesthat the protein includes polypeptide components (i e., amino acidsubsequences) derived from more than one parental viral strain orsource. As will be apparent herein, such chimeric viruses are typicallyreassortant/recombinant viruses. Thus, in some embodiments, a chimeracan optionally include, e.g., a sequence (e.g., of HA and/or NA) from anA influenza virus placed into a backbone comprised of, orconstructed/derived from a B influenza virus (e.g., B/AA/1/66, etc.) ora B influenza virus sequence placed into an A influenza virus backbone(i.e., donor virus) such as, e.g., A/AA/6/60, etc.

The term “recombinant” indicates that the material (e.g., a nucleic acidor protein) has been artificially or synthetically (non-naturally)altered by human intervention. The alteration can be performed on thematerial within, or removed from, its natural environment or state.Specifically, e.g., an influenza virus is recombinant when it isproduced by the expression of a recombinant nucleic acid. For example, a“recombinant nucleic acid” is one that is made by recombining nucleicacids, e.g., during cloning, DNA shuffling or other procedures, or bychemical or other mutagenesis; a “recombinant polypeptide” or“recombinant protein” is a polypeptide or protein which is produced byexpression of a recombinant nucleic acid; and a “recombinant virus,”e.g., a recombinant influenza virus, is produced by the expression of arecombinant nucleic acid.

The term “reassortant,” when referring to a virus (typically herein, aninfluenza virus), indicates that the virus includes genetic and/orpolypeptide components derived from more than one parental viral strainor source. For example, a 7:1 reassortant includes 7 viral genomicsegments (or gene segments) derived from a first parental virus, and asingle complementary viral genomic segment, e.g., encoding ahemagglutinin or neuraminidase such as those listed in the SEQ ID Tablesherein (e.g., SEQ ID NO: 1-96). A 6:2 reassortant includes 6 genomicsegments, most commonly the 6 internal genes from a first parentalvirus, and two complementary segments, e.g., hemagglutinin andneuraminidase, from one or more different parental virus. Reassortantviruses can also, depending upon context herein, be termed as “chimeric”and/or “recombinant.”

The term “introduced” when referring to a heterologous or isolatednucleic acid refers to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid can beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA). The term includes suchmethods as “infection,” “transfection,” “transformation,” and“transduction.” In the context of the invention a variety of methods canbe employed to introduce nucleic acids into cells, includingelectroporation, calcium phosphate precipitation, lipid mediatedtransfection (lipofection), etc.

The term “host cell” means a cell that contains a heterologous nucleicacid, such as a vector or a virus, and supports the replication and/orexpression of the nucleic acid. Host cells can be prokaryotic cells suchas E. coli, or eukaryotic cells such as yeast, insect, amphibian, avianor mammalian cells, including human cells. Exemplary host cells caninclude, e.g., Vero (African green monkey kidney) cells, BHK (babyhamster kidney) cells, primary chick kidney (PCK) cells, Madin-DarbyCanine Kidney (MDCK) cells, Madin-Darby Bovine Kidney (MDBK) cells, 293cells (e.g., 293T cells), and COS cells (e.g., COS1, COS7 cells), etc.In other embodiments, host cells can optionally include eggs (e.g., heneggs, embryonated hen eggs, etc.).

An “immunologically effective amount” of influenza virus is an amountsufficient to enhance an individual's (e.g., a human's) own immuneresponse against a subsequent exposure to influenza virus. Levels ofinduced immunity can be monitored, e.g., by measuring amounts ofneutralizing secretory and/or serum antibodies, e.g., by plaqueneutralization, complement fixation, enzyme-linked immunosorbent, ormicroneutralization assay.

A “protective immune response” against influenza virus refers to animmune response exhibited by an individual (e.g., a human) that isprotective against disease when the individual is subsequently exposedto and/or infected with wild-type influenza virus. In some instances,the wild-type (e.g., naturally circulating) influenza virus can stillcause infection, but it cannot cause a serious or life-threateninginfection. Typically, the protective immune response results indetectable levels of host engendered serum and secretory antibodies thatare capable of neutralizing virus of the same strain and/or subgroup(and possibly also of a different, non-vaccine strain and/or subgroup)in vitro and in vivo.

As used herein, an “antibody” is a protein comprising one or morepolypeptides substantially or partially encoded by immunoglobulin genesor fragments of immunoglobulin genes. The recognized immunoglobulingenes include the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes, as well as myriad immunoglobulin variable regiongenes. Light chains are classified as either kappa or lambda. Heavychains are classified as gamma, mu, alpha, delta, or epsilon, which inturn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,respectively. A typical immunoglobulin (antibody) structural unitcomprises a tetramer. Each tetramer is composed of two identical pairsof polypeptide chains, each pair having one “light” (about 25 kD) andone “heavy” chain (about 50-70 kD). The N-terminus of each chain definesa variable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain (VL)and variable heavy chain (VH) refer to these light and heavy chainsrespectively. Antibodies exist as intact immunoglobulins or as a numberof well-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′2, a dimer ofFab which itself is a light chain joined to VH-CH1 by a disulfide bond.The F(ab)′2 may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)2 dimer into aFab′ monomer. The Fab′ monomer is essentially a Fab with part of thehinge region (see Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1999) for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein, includes antibodies or fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Antibodies include, e.g., polyclonalantibodies, monoclonal antibodies, multiple or single chain antibodies,including single chain Fv (sFv or scFv) antibodies in which a variableheavy and a variable light chain are joined together (directly orthrough a peptide linker) to form a continuous polypeptide, andhumanized or chimeric antibodies.

Influenza Virus

The polypeptides and polynucleotides of the invention are variants ofinfluenza HA and/or NA sequences. See, e.g., the Sequence Listing inFIGS. 1 and 2 below. In general, influenza viruses are made up of aninternal ribonucleoprotein core containing a segmented single-strandedRNA genome and an outer lipoprotein envelope lined by a matrix protein.The genome of influenza viruses is composed of eight segments of linear(−) strand ribonucleic acid (RNA), encoding the immunogenichemagglutinin (HA) and neuraminidase (NA) proteins, and six internalcore polypeptides: the nucleocapsid nucleoprotein (NP); matrix proteins(M); non-structural proteins (NS); and 3 RNA polymerase (PA, PB1, PB2)proteins. During replication, the genomic viral RNA is transcribed into(+) strand messenger RNA and (−) strand genomic cRNA in the nucleus ofthe host cell. Each of the eight genomic segments is packaged intoribonucleoprotein complexes that contain, in addition to the RNA, NP anda polymerase complex (PB1, PB2, and PA). The hemagglutinin moleculeconsists of a surface glycoprotein and acts to bind toN-AcetylNeuraminic acid (NeuNAc), also known as sialic acid, on hostcell surface receptors. In some embodiments herein, the polypeptides ofthe invention (and polypeptides encoded by the polynucleotides of theinvention) can act to bind NeuNAc whether in vitro or in vivo. Suchaction can in some embodiments also be done by fragments ofhemagglutinin which retain hemagglutinin activity. Hemagglutinin is madeup of two subunits, HA1 and HA2 and the entire structure is about 550amino acids in length and about 220 kD. Neuraminidase molecules cleaveterminal sialic acid residues from cell surface receptors of influenzavirus, thereby releasing virions from infected cells. Neuraminidase alsoremoves sialic acid from newly made hemagglutinin and neuraminidasemolecules. In some embodiments herein, the polypeptides of the invention(and polypeptides encoded by the polynucleotides of the invention) canact to cleave sialic acid residues whether in vitro or in vivo. Thisaction can also be done in some embodiments by fragments ofneuraminidase which retain neuraminidase activity. The neuraminidasepolypeptides of the invention show immunological cross reactivity withone or more known neuraminidase molecule from an influenza virus. Theliterature is replete with examples of such known neuraminidases (e.g.,in GenBank, in publications from the CDC, etc.). Similarly, thehemagglutinin polypeptides of the invention show immunologicalcross-reactivity with one or more known hemagglutinin molecule from aninfluenza virus. Again, the literature is replete with examples of suchknown hemagglutinin molecules.

Influenza is commonly grouped into influenza A and influenza Bcategories, as well as a typically less important C category. InfluenzaA and influenza B viruses each contain eight segments of single strandedRNA with negative polarity. The influenza A genome encodes elevenpolypeptides. Segments 1-3 encode three polypeptides, making up aRNA-dependent RNA polymerase. Segment 1 encodes the polymerase complexprotein PB2. The remaining polymerase proteins PB1 and PA are encoded bysegment 2 and segment 3, respectively. In addition, segment 1 of someinfluenza strains encodes a small protein, PB1-F2, produced from analternative reading frame within the PB1 coding region. Segment 4encodes the hemagglutinin (HA) surface glycoprotein involved in cellattachment and entry during infection. Segment 5 encodes thenucleocapsid nucleoprotein (NP) polypeptide, the major structuralcomponent associated with viral RNA. Segment 6 encodes a neuraminidase(NA) envelope glycoprotein. Segment 7 encodes two matrix proteins,designated M1 and M2, which are translated from differentially splicedmRNAs. Segment 8 encodes NS1 and NS2, two nonstructural proteins, whichare translated from alternatively spliced mRNA variants. The eightgenome segments of influenza B encode 11 proteins. The three largestgenes code for components of the RNA polymerase, PB1, PB2 and PA.Segment 4 encodes the HA protein. Segment 5 encodes NP. Segment 6encodes the NA protein and the NB protein. Both proteins, NB and NA, aretranslated from overlapping reading frames of a bicistronic mRNA.Segment 7 of influenza B also encodes two proteins: M1 and BM2. Thesmallest segment encodes two products: NS1 is translated from the fulllength RNA, while NS2 is translated from a spliced mRNA variant.

Influenza types A and B are typically associated with influenzaoutbreaks in human populations. However, type A influenza also infectsother creatures as well, e.g., birds, pigs, and other animals. The typeA viruses are categorized into subtypes based upon differences withintheir hemagglutinin and neuraminidase surface glycoprotein antigens.Hemagglutinin in type A viruses has 14 known subtypes and neuraminidasehas 9 known subtypes. In humans, currently only about 3 differenthemagglutinin and 2 different neuraminidase subtypes are known, e.g.,H1, H2, H3, N1, and N2. In particular, two major subtypes of influenza Ahave been active in humans, namely, H1N1 and H3N2. H1N2, however hasrecently been of concern. Influenza B viruses are not divided intosubtypes based upon their hemagglutinin and neuraminidase proteins. Aswill be appreciated, the sequences contained within the sequence listingin FIG. 1 comprise a number of different subtypes of influenza. Thus,for example in the sequence listing A-H3N2 strains are exampled by caA/Shandong/9/93, ca A/Johannesburg/33/94-like, ca A/Wuhan/395/95, caA/Sydney/05/97, ca A/Panama/2007/99, ca A/Wyoming/03/2003. A-H1N1strains are shown in ca A/Texas/36/91, ca A/Shenzhen/227/95, caA/Beijing/262/95, and ca A/New Calcdonia/20/99, while B-HANA strainsinclude ca B/Ann Arbor/1/94, ca B/Yamanashi/166/98, caB/Johannesburg/5/99, ca B/Victoria/504/2000, ca B/Hong Kong/330/2001, caB/Brisbane/32/2002, and ca B/Jilin/20/2003, etc. The Figures also showthe subtypes of the other specific strains as well. As can be seen fromthe Figures several sequences are A/Fujian-like strains (e.g., caA/Wellington/01/2004 (for classical reassorted), caA/Malaysia/1/2004_(—)1 (186G, 193R in HA) (for plasmid-derived, caA/Malaysia/1/2004_(—)2 (186V, 193S in HA) (for both plasmid-derived andclassical reassorted). Other sequences are ca B/Shanghai-like strains(e.g., ca B/Jiangshu/10/2003 (for both classical reassorted andplasmid-derived), ca B/Shanghai/361/2002 (for plasmid-derived).

Different strains of influenza can be categorized based upon, e.g., theability of influenza to agglutinate red blood cells (RBCs orerythrocytes). Antibodies specific for particular influenza strains canbind to the virus and, thus, prevent such agglutination. Assaysdetermining strain types based on such inhibition are typically known ashemagglutinin inhibition assays (HI assays or HAI assays) and arestandard and well known methods in the art to characterize influenzastrains. Of course, those of skill in the art will be familiar withother assays, e.g., ELISA, indirect fluorescent antibody assays,immunohistochemistry, Western blot assays, etc. with which tocharacterize influenza strains and the use of and discussion herein ofHI assays should not be necessarily construed as limiting.

Briefly, in typical HI assays, sera to be used for typing orcategorization, which is often produced in ferrets, is added toerythrocyte samples in various dilutions, e.g., 2-fold, etc. Opticaldetermination is then made whether the erythrocytes are clumped together(i.e., agglutinated) or are suspended (i.e., non-agglutinated). If thecells are not clumped, then agglutination did not occur due to theinhibition from antibodies in the sera that are specific for thatinfluenza. Thus, the types of influenza are defined as being within thesame strain. In some cases, one strain is described as being “like” theother, e.g., strain x is a “y-like” strain, etc. For example, if twosamples are within four-fold titer of one another as measured by an HIassay, then they can be described as belonging to the same strain (e.g.,both belonging to the “New Calcdonia” strain or both being “Moscow-like”strains, etc.). In other words, strains are typically categorized basedupon their immunologic or antigenic profile. An HAI titer is typicallydefined as the highest dilution of a serum that completely inhibitshemagglutination. See, e.g., Schild, et al., Bull. Wld Hlth Org., 1973,48:269-278, etc. Again, those of skill in the art will be quite familiarwith categorization and classification of influenza into strains and themethods to do so.

From the above it will be appreciated that the current invention notonly comprises the specific sequences listed herein, but also suchsequences within various vectors (e.g., ones used for plasmidreassortment and rescue, see below) as well as hemagglutinin andneuraminidase sequences within the same strains as the sequences listedherein. Also, such same strains that are within various vectors (e.g.,typically ones used for plasmid reassortment and rescue such as A/AnnArbor/6/60 or B/Ann Arbor/1/66, A/Puerto Rico/8/34,B/Leningrad/14/17/55, B/14/5/1, B/USSR/60/69, B/Leningrad/179/86,B/Leningrad/14/55, or B/England/2608/76, etc.) are also included.

As used herein, the term “similar strain” should be taken to indicatethat a first influenza virus is of the same or related strain as asecond influenza virus. In typical embodiments such relation is commonlydetermined through use of an HAI assay. Influenza viruses that fallwithin a four-fold titer of one another in an HAI assay are, thus, of a“similar strain.” Those of skill in the art, however, will be familiarwith other assays, etc. to determine similar strains, e.g., FRID,neutralization assays, etc. The current invention also comprises suchsimilar strains (i.e., strains similar to the ones present in thesequence listing herein) in the various plasmids, vectors, viruses,methods, etc. herein. Thus, unless the context clearly dictatesotherwise, descriptions herein of particular sequences (e.g., those inthe sequence listing) or fragments thereof also should be considered toinclude sequences from similar strains to those (i.e., similar strainsto those strains having the sequences in those plasmids, vectors,viruses, etc. herein). Also, it will be appreciated that the NA and HApolypeptides within such similar strains are, thus, “similarpolypeptides” when compared between “similar strains.”

Influenza Virus Vaccines

The sequences, compositions and methods herein are primarily, but notsolely, concerned with production of influenza viruses for vaccines.Historically, influenza virus vaccines have primarily been produced inembryonated hen eggs using strains of virus selected or based onempirical predictions of relevant strains. More recently, reassortantviruses have been produced that incorporate selected hemagglutininand/or neuraminidase antigens in the context of an approved attenuated,temperature sensitive master strain. Following culture of the virusthrough multiple passages in hen eggs, influenza viruses are recoveredand, optionally, inactivated, e.g., using formaldehyde and/orβ-propiolactone (or alternatively used in live attenuated vaccines).Thus, it will be appreciated that HA and NA sequences (as in the currentinvention) are quite useful in constructing influenza vaccines.

Attempts at producing recombinant and reassortant vaccines in cellculture have been hampered by the inability of some of the strainsapproved for vaccine production to grow efficiently under standard cellculture conditions. However, prior work by the inventors and theircoworkers provided a vector system, and methods for producingrecombinant and reassortant viruses in culture, thus, making it possibleto rapidly produce vaccines corresponding to one or many selectedantigenic strains of virus, e.g., either A or B strains, varioussubtypes or substrains, etc., e.g., comprising the HA and NA sequencesherein. See, U.S. Application No. 60/420,708, filed Oct. 23, 2002, U.S.application Ser. No. 10/423,828, filed Apr. 25, 2003, and U.S.Application No. 60/574,117, filed May 24, 2004, all entitled“Multi-Plasmid System for the Production of Influenza Virus.” Typically,the cultures are maintained in a system, such as a cell cultureincubator, under controlled humidity and CO₂, at constant temperatureusing a temperature regulator, such as a thermostat to insure that thetemperature does not exceed 35° C. Reassortant influenza viruses can bereadily obtained by introducing a subset of vectors corresponding togenomic segments of a master influenza virus, in combination withcomplementary segments derived from strains of interest (e.g., HA and NAantigenic variants herein). Typically, the master strains are selectedon the basis of desirable properties relevant to vaccine administration.For example, for vaccine production, e.g., for production of a liveattenuated vaccine, the master donor virus strain may be selected for anattenuated phenotype, cold adaptation and/or temperature sensitivity. Asexplained elsewhere herein and, e.g., in U.S. patent application Ser.No. 10/423,828, etc., various embodiments of the invention utilize A/AnnArbor (AA)/6/60 or B/Ann Arbor/1/66 or A/Puerto Rico/8/34, orB/Leningrad/14/17/55, B/14/5/1, B/USSR/60/69, B/Leningrad/179/86,B/Leningrad/14/55, or B/England/2608/76 influenza strain as a “backbone”upon which to add HA and/or NA genes (e.g., such as those sequenceslisted herein, etc.) to create desired reassortant viruses. Thus, forexample, in a 6:2 reassortant, 2 genes (i.e., NA and HA) would be fromthe influenza strain(s) against which an immunogenic reaction isdesired, while the other 6 genes would be from the Ann Arbor strain, orother backbone strain, etc. The Ann Arbor virus is useful for its coldadapted, attenuated, temperature sensitive attributes. Of course, itwill be appreciated that the HA and NA sequences herein are capable ofreassortment with a number of other virus genes or virus types (e.g., anumber of different “backbones” such as A/Puerto Rico/8/34, etc.,containing the other influenza genes present in a reassortant, namely,the non-HA and non-NA genes). Live, attenuated influenza A virusvaccines against human influenza viruses were recently licensed in theUnited States. See above. Such vaccines are reassortant H1N1 and H1N2viruses in which the internal protein genes of A/Ann Arbor (AA)/6/60(H2N2) cold adapted (ca) virus confer the cold adapted, attenuation andtemperature sensitive phenotypes of the AA ca virus on the reassortantviruses (i.e., the ones having the hemagglutinin and neuraminidase genesfrom the non-Ann Arbor strain). In some embodiments herein, thereassortants can also comprise 7:1 reassortants. In other words, onlythe HA or the NA is not from the backbone or MDV strain. Previous workhas been reported with suitable backbone donor virus strains thatoptionally are within various embodiments of the current invention. See,e.g., U.S. Application No. 60/420,708, filed Oct. 23, 2002, U.S.application Ser. No. 10/423,828, filed Apr. 25, 2003, and U.S.Application No. 60/574,117, filed May 25, 2004, all entitled“Multi-Plasmid System for the Production of Influenza Virus”; Maassab etal., J. of Inf. Dis., 1982, 146:780-790; Cox, et al., Virology, 1988,167:554-567; Wareing et al., Vaccine, 2001, 19:3320-3330; Clements, etal., J Infect Dis., 1990, 161(5):869-77, etc.

In some embodiments, the sequences herein can optionally have specificregions removed (both or either in the nucleic acid sequence or theamino acid sequence). For example, for those molecules having apolybasic cleavage site, such sites can optionally be removed. Suchcleavage sites, in some embodiments herein, are, e.g., modified oraltered in their sequences in comparison to the wild-type sequences fromwhich such sequences are derived (e.g., to disable the cleavage orreduce the cleavage there, etc.). Such modifications/alterations can bedifferent in different strains or sequences due to the various sequencesof the cleavage sites in the starting sequences. For example, 4polybasic residues (RRKK) are typically removed in some HA sequences.(as compared to wt). In various embodiments, such polybasic cleavagesites can be modified in a number of ways (all of which are containedwithin the invention). For example, the polybasic cleavage site can beremoved one amino acid at a time (e.g., one R removed, two Rs removed,RRK removed, or RRKK removed). Additionally, an amino acid residuedirectly upstream of the cleavage site can also be removed or altered(e.g., from an R to a T, etc.); also, the nucleotides encoding the aminoacid residue directly after the cleavage site can also be modified.Those of skill in the art will be familiar with various methods ofremoving such specific regions. The resulting shortened sequences arealso contained within the current invention. See, e.g., Li et al., J. ofInfectious Diseases, 179:1132-8, 1999

The terms “temperature sensitive,” “cold adapted” and “attenuated” asapplied to viruses (typically used as vaccines or for vaccineproduction) which optionally encompass the current sequences, are wellknown in the art. For example, the term “temperature sensitive” (ts)indicates, e.g., that the virus exhibits a 100 fold or greater reductionin titer at 39° C. relative to 33° C. for influenza A strains, or thatthe virus exhibits a 100 fold or greater reduction in titer at 37° C.relative to 33° C. for influenza B strains. The term “cold adapted” (ca)indicates that the virus exhibits growth at 25° C. within 100 fold ofits growth at 33° C., while the term “attenuated” (au) indicates thatthe virus replicates in the upper airways of ferrets but is notdetectable in their lung tissues, and does not cause influenza-likeillness in the animal. It will be understood that viruses withintermediate phenotypes, i.e., viruses exhibiting titer reductions lessthan 100 fold at 39° C. (for A strain viruses) or 37° C. (for B strainviruses), or exhibiting growth at 25° C. that is more than 100 fold thanits growth at 33° C. (e.g., within 200 fold, 500 fold, 1000 fold, 10,000fold less), and/or exhibit reduced growth in the lungs relative togrowth in the upper airways of ferrets (i.e., partially attenuated)and/or reduced influenza like illness in the animal, are also usefulviruses and can be used in conjunction with the HA and NA sequencesherein.

Thus, the present invention can utilize growth, e.g., in appropriateculture conditions, of virus strains (both A strain and B straininfluenza viruses) with desirable properties relative to vaccineproduction (e.g., attenuated pathogenicity or phenotype, coldadaptation, temperature sensitivity, etc.) in vitro in cultured cells.Influenza viruses can be produced by introducing a plurality of vectorsincorporating cloned viral genome segments into host cells, andculturing the cells at a temperature not exceeding 35° C. When vectorsincluding an influenza virus genome are transfected, recombinant virusessuitable as vaccines can be recovered by standard purificationprocedures. Using the vector system and methods of the invention,reassortant viruses incorporating the six internal gene segments of astrain selected for its desirable properties with respect to vaccineproduction, and the immunogenic HA and NA segments from a selected,e.g., pathogenic strain such as those in the sequence listing herein,can be rapidly and efficiently produced in tissue culture. Thus, thesystem and methods described herein are useful for the rapid productionin cell culture of recombinant and reassortant influenza A and Bviruses, including viruses suitable for use as vaccines, including liveattenuated vaccines, such as vaccines suitable for intranasaladministration.

In such embodiments, typically, a single Master Donor Virus (MDV) strainis selected for each of the A and B subtypes. In the case of a liveattenuated vaccine, the Master Donor Virus strain is typically chosenfor its favorable properties, e.g., temperature sensitivity, coldadaptation and/or attenuation, relative to vaccine production. Forexample, exemplary Master Donor Strains include such temperaturesensitive, attenuated and cold adapted strains of A/Ann Arbor/6/60 andB/Ann Arbor/1/66, respectively, as well as others mentioned throughout.

For example, a selected master donor type A virus (MDV-A), or masterdonor type B virus (MDV-B), is produced from a plurality of cloned viralcDNAs constituting the viral genome. Embodiments include those whereinrecombinant viruses are produced from eight cloned viral cDNAs. Eightviral cDNAs representing either the selected MDV-A or MDV-B sequences ofPB2, PB1, PA, NP, HA, NA, M and NS are optionally cloned into abi-directional expression vector, such as a plasmid (e.g., pAD3000),such that the viral genomic RNA can be transcribed from an RNApolymerase I (pol I) promoter from one strand and the viral mRNAs can besynthesized from an RNA polymerase II (pol II) promoter from the otherstrand. Optionally, any gene segment can be modified, including the HAsegment (e.g., to remove the multi-basic cleavage site (also known as apolybasic cleavage site)).

Infectious recombinant MDV-A or MDV-B virus can be then recoveredfollowing transfection of plasmids bearing the eight viral cDNAs intoappropriate host cells, e.g., Vero cells, co-cultured MDCK/293T orMDCK/COS7 cells. Using the plasmids and methods described herein and,e.g., in U.S. Application No. 60/420,708, filed Oct. 23, 2002, U.S.application Ser. No. 10/423,828, filed Apr. 25, 2003, and U.S.Application No. 60/574,117, filed May 24, 2004, all entitled“Multi-Plasmid System for the Production of Influenza Virus”; Hoffmann,E., 2000, PNAS, 97(11):6108-6113; U.S. Published Patent Application No.20020164770 to Hoffmann; and U.S. Pat. No. 6,544,785 issued Apr. 8, 2003to Palese, et al., the invention is useful, e.g., for generating 6:2reassortant influenza vaccines by co-transfection of the 6 internalgenes (PB1, PB2, PA, NP, M and NS) of the selected virus (e.g., MDV-A,MDV-B) together with the HA and NA derived from different correspondingtype (A or B) influenza viruses e.g., as shown in the sequence listingsherein. For example, the HA segment is favorably selected from apathogenically relevant H1, H3 or B strain, as is routinely performedfor vaccine production. Similarly, the HA segment can be selected from astrain with emerging relevance as a pathogenic strain such as those inthe sequence listing herein. Reassortants incorporating seven genomesegments of the MDV and either the HA or NA gene of a selected strain(7:1 reassortants) can also be produced. It will be appreciated, and asis detailed throughout, the molecules of the invention can optionally becombined in any desired combination. For example, the HA and/or NAsequences herein can be placed, e.g., into a reassortant backbone suchas A/AA/6/60, B/AA/1/66, A/Puerto Rico/8/34 (i.e., PR8), etc., in 6:2reassortants or 7:1 reassortants, etc. Thus, as explained more fullybelow, there would be 6 backbone gene regions from the donor virus(again, e.g., A/AA/6/60, etc.) and 2 genes regions from a second strain(e.g., a wild-type strain, not the backbone donor virus). Such 2 generegions are preferably the HA and NA genes. A similar situation arisesfor 7:1 reassortants, in which however, there are 7 gene regions fromthe background donor virus and 1 gene (either HA or NA) from a differentvirus (typically wild-type or one to which an immune response isdesired). Also, it will be appreciated that the sequences herein (e.g.,those in the sequence listing of FIG. 1, etc.) can be combined in anumber of means in different embodiments herein. Thus, any of thesequences herein can be present singularly in a 7:1 reassortant (i.e.,the sequence of the invention present with 7 backbone donor virus generegions) and/or can be present with another sequence of the invention ina 6:2 reassortant. Within such 6:2 reassortants, any of the sequences ofthe invention can optionally be present with any other sequence of theinvention. Typical, and preferred, embodiments comprise HA and NA fromthe same original wild-type strains however (or modified wild-typestrains such as those with modified polybasic cleavage sites). Forexample, typical embodiments can comprise a 6:2 reassortant having 6gene regions from a backbone donor virus such as A/AA/6/60 and the HAand NA gene regions from the same strain such as ca A/Shandong/9/93 orboth HA and NA from ca A/Wuhan/395/95 or both HA and NA from ca B/AnnArbor/1/94 (which would typically, but not exclusively, be presentwithin a B influenza backbone donor virus such as B/Ann Arbor/1/66,etc.), etc. Of course, it will again be appreciated that the inventionalso includes such reassortant viruses wherein the non-background generegions (i.e., the HA and/or NA regions) are from similar strains (i.e.,strains that are similar strains to influenza strains having thesequences found in SEQ ID NO:1-48. The above references are specificallyincorporated herein in their entirety for all purposes, e.g., especiallyfor their teachings regarding plasmids, plasmid rescue of virus(influenza virus), multi-plasmid systems for virus rescue/production,etc.

Again, the HA and NA sequences of the current invention are optionallyutilized in such plasmid reassortment vaccines (and/or in other ts, cs,ca, and/or att viruses and vaccines). However, it should be noted thatthe HA and NA sequences, etc. of the invention are not limited tospecific vaccine compositions or production methods, and can, thus, beutilized in substantially any vaccine type or vaccine production methodwhich utilizes strain specific HA and NA antigens (e.g., the sequencesof the invention).

Flumist™

As mentioned previously, numerous examples and types of influenzavaccine exist. An exemplary influenza vaccine is FluMist™ (MedImmuneVaccines Inc., Mt. View, Calif.) which is a live, attenuated vaccinethat protects children and adults from influenza illness (Belshe et al.(1998) The efficacy of live attenuated, cold-adapted, trivalent,intranasal influenza virus vaccine in children N Engl J Med 338:1405-12;Nichol et al. (1999) Effectiveness of live, attenuated intranasalinfluenza virus vaccine in healthy, working adults: a randomizedcontrolled trial JAMA 282:137-44). In typical, and preferred,embodiments, the methods and compositions of the current invention arepreferably adapted to/used with production of FluMist™ vaccine. However,it will be appreciated by those skilled in the art that the sequences,methods, compositions, etc. herein are also adaptable to production ofsimilar or even different viral vaccines.

FluMist™ vaccine strains contain, e.g., HA and NA gene segments derivedfrom the wild-type strains to which the vaccine is addressed (or, insome instances, to related strains) along with six gene segments, PB1,PB2, PA, NP, M and NS, from a common master donor virus (MDV). The HAand NA sequences herein, thus, are optionally part of various FluMist™formulations. The MDV for influenza A strains of FluMist™ (MDV-A), wascreated by serial passage of the wild-type A/Ann Arbor/6/60 (A/AA/6/60)strain in primary chicken kidney tissue culture at successively lowertemperatures (Maassab (1967) Adaptation and growth characteristics ofinfluenza virus at 25 degrees C. Nature 213:612-4). MDV-A replicatesefficiently at 25° C. (ca, cold adapted), but its growth is restrictedat 38 and 39° C. (ts, temperature sensitive). Additionally, this virusdoes not replicate in the lungs of infected ferrets (att, attenuation).The ts phenotype is believed to contribute to the attenuation of thevaccine in humans by restricting its replication in all but the coolestregions of the respiratory tract. The stability of this property hasbeen demonstrated in animal models and clinical studies. In contrast tothe ts phenotype of influenza strains created by chemical mutagenesis,the ts property of MDV-A does not revert following passage throughinfected hamsters or in shed isolates from children (for a recentreview, see Murphy & Coelingh (2002) Principles underlying thedevelopment and use of live attenuated cold-adapted influenza A and Bvirus vaccines Viral Immunol 15:295-323).

Clinical studies in over 20,000 adults and children involving 12separate 6:2 reassortant strains have shown that these vaccines areattenuated, safe and efficacious (Belshe et al. (1998) The efficacy oflive attenuated, cold-adapted, trivalent, intranasal influenza virusvaccine in children N Engl J Med 338:1405-12; Boyce et al. (2000) Safetyand immunogenicity of adjuvanted and unadjuvanted subunit influenzavaccines administered intranasally to healthy adults Vaccine 19:217-26;Edwards et al. (1994) A randomized controlled trial of cold adapted andinactivated vaccines for the prevention of influenza A disease J InfectDis 169:68-76; Nichol et al. (1999) Effectiveness of live, attenuatedintranasal influenza virus vaccine in healthy, working adults: arandomized controlled trial JAMA 282:137-44). Reassortants carrying thesix internal genes of MDV-A and the two HA and NA gene segments of awild-type virus (i.e., a 6:2 reassortant) consistently maintain ca, tsand att phenotypes (Maassab et al. (1982) Evaluation of acold-recombinant influenza virus vaccine in ferrets J. Infect. Dis.146:780-900).

Production of such reassorted virus using B strains of influenza is moredifficult, however, recent work (see, e.g., U.S. Application No.60/420,708, filed Oct. 23, 2002, U.S. application Ser. No. 10/423,828,filed Apr. 25, 2003, and U.S. Application No. 60/574,117, filed May 24,2004, all entitled “Multi-Plasmid System for the Production of InfluenzaVirus”) has shown an eight plasmid system for the generation ofinfluenza B virus entirely from cloned cDNA. Methods for the productionof attenuated live influenza A and B virus suitable for vaccineformulations, such as live virus vaccine formulations useful forintranasal administration were also shown.

The system and methods described previously are useful for the rapidproduction in cell culture of recombinant and reassortant influenza Aand B viruses, including viruses suitable for use as vaccines, includinglive attenuated vaccines, such as vaccines suitable for intranasaladministration. The sequences, methods, etc. of the current invention,are optionally used in conjunction with, or in combination with, suchprevious work involving, e.g., reassorted influenza viruses for vaccineproduction to produce viruses for vaccines.

Methods and Compositions for Prophylactic Administration of Vaccines

As stated above, alternatively, or in addition to, use in production ofFluMist™ vaccine, the current invention can be used in other vaccineformulations. In general, recombinant and reassortant viruses of theinvention (e.g., those comprising polynucleotides of SEQ ID NO:1-48 orpolypeptides of SEQ ID NO:49-96, or similar strains of the virussequences within SEQ ID NO:1-96, or fragments of any of the previous)can be administered prophylactically in an immunologically effectiveamount and in an appropriate carrier or excipient to stimulate an immuneresponse specific for one or more strains of influenza virus asdetermined by the HA and/or NA sequence. Typically, the carrier orexcipient is a pharmaceutically acceptable carrier or excipient, such assterile water, aqueous saline solution, aqueous buffered salinesolutions, aqueous dextrose solutions, aqueous glycerol solutions,ethanol, allantoic fluid from uninfected hen eggs (i.e., normalallantoic fluid or NAF), or combinations thereof. The preparation ofsuch solutions insuring sterility, pH, isotonicity, and stability iseffected according to protocols established in the art. Generally, acarrier or excipient is selected to minimize allergic and otherundesirable effects, and to suit the particular route of administration,e.g., subcutaneous, intramuscular, intranasal, etc.

A related aspect of the invention provides methods for stimulating theimmune system of an individual to produce a protective immune responseagainst influenza virus. In the methods, an immunologically effectiveamount of a recombinant influenza virus (e.g., an HA and/or an NAmolecule of the invention), an immunologically effective amount of apolypeptide of the invention, and/or an immunologically effective amountof a nucleic acid of the invention is administered to the individual ina physiologically acceptable carrier.

Generally, the influenza viruses of the invention are administered in aquantity sufficient to stimulate an immune response specific for one ormore strains of influenza virus (i.e., against the HA and/or NA strainsof the invention). Preferably, administration of the influenza viruseselicits a protective immune response to such strains. Dosages andmethods for eliciting a protective immune response against one or moreinfluenza strains are known to those of skill in the art. See, e.g.,U.S. Pat. No. 5,922,326; Wright et al., Infect. Immun. 37:397-400(1982); Kim et al., Pediatrics 52:56-63 (1973); and Wright et al., J.Pediatr. 88:931-936 (1976). For example, influenza viruses are providedin the range of about 1-1000 HID₅₀ (human infectious dose), i.e., about10⁵-10⁸ pfu (plaque forming units) per dose administered. Typically, thedose will be adjusted within this range based on, e.g., age, physicalcondition, body weight, sex, diet, time of administration, and otherclinical factors. The prophylactic vaccine formulation is systemicallyadministered, e.g., by subcutaneous or intramuscular injection using aneedle and syringe, or a needle-less injection device. Alternatively,the vaccine formulation is administered intranasally, either by drops,large particle aerosol (greater than about 10 microns), or spray intothe upper respiratory tract. While any of the above routes of deliveryresults in a protective systemic immune response, intranasaladministration confers the added benefit of eliciting mucosal immunityat the site of entry of the influenza virus. For intranasaladministration, attenuated live virus vaccines are often preferred,e.g., an attenuated, cold adapted and/or temperature sensitiverecombinant or reassortant influenza virus. See above. While stimulationof a protective immune response with a single dose is preferred,additional dosages can be administered, by the same or different route,to achieve the desired prophylactic effect.

Typically, the attenuated recombinant influenza of this invention asused in a vaccine is sufficiently attenuated such that symptoms ofinfection, or at least symptoms of serious infection, will not occur inmost individuals immunized (or otherwise infected) with the attenuatedinfluenza virus. In some instances, the attenuated influenza virus canstill be capable of producing symptoms of mild illness (e.g., mild upperrespiratory illness) and/or of dissemination to unvaccinatedindividuals. However, its virulence is sufficiently abrogated such thatsevere lower respiratory tract infections do not occur in the vaccinatedor incidental host.

Alternatively, an immune response can be stimulated by ex vivo or invivo targeting of dendritic cells with influenza viruses comprising thesequences herein. For example, proliferating dendritic cells are exposedto viruses in a sufficient amount and for a sufficient period of time topermit capture of the influenza antigens by the dendritic cells. Thecells are then transferred into a subject to be vaccinated by standardintravenous transplantation methods.

While stimulation of a protective immune response with a single dose ispreferred, additional dosages can be administered, by the same ordifferent route, to achieve the desired prophylactic effect. In neonatesand infants, for example, multiple administrations may be required toelicit sufficient levels of immunity. Administration can continue atintervals throughout childhood, as necessary to maintain sufficientlevels of protection against wild-type influenza infection. Similarly,adults who are particularly susceptible to repeated or serious influenzainfection, such as, for example, health care workers, day care workers,family members of young children, the elderly, and individuals withcompromised cardiopulmonary function may require multiple immunizationsto establish and/or maintain protective immune responses. Levels ofinduced immunity can be monitored, for example, by measuring amounts ofneutralizing secretory and serum antibodies, and dosages adjusted orvaccinations repeated as necessary to elicit and maintain desired levelsof protection.

Optionally, the formulation for prophylactic administration of theinfluenza viruses also contains one or more adjuvants for enhancing theimmune response to the influenza antigens. Suitable adjuvants include:complete Freund's adjuvant, incomplete Freund's adjuvant, saponin,mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil orhydrocarbon emulsions, bacille Calmette-Guerin (BCG), Corynebacteriumparvum, and the synthetic adjuvants QS-21 and MF59.

If desired, prophylactic vaccine administration of influenza viruses canbe performed in conjunction with administration of one or moreimmunostimulatory molecules Immunostimulatory molecules include variouscytokines, lymphokines and chemokines with immunostimulatory,immunopotentiating, and pro-inflammatory activities, such asinterleukins (e.g., IL-1, IL-2, IL-3, IL-4, IL-12, IL-13); growthfactors (e.g., granulocyte-macrophage (GM)-colony stimulating factor(CSF)); and other immunostimulatory molecules, such as macrophageinflammatory factor, Flt3 ligand, B7.1; B7.2, etc. The immunostimulatorymolecules can be administered in the same formulation as the influenzaviruses, or can be administered separately. Either the protein (e.g., anHA and/or NA polypeptide of the invention) or an expression vectorencoding the protein can be administered to produce an immunostimulatoryeffect.

The above described methods are useful for therapeutically and/orprophylactically treating a disease or disorder, typically influenza, byintroducing a vector of the invention comprising a heterologouspolynucleotide encoding a therapeutically or prophylactically effectiveHA and/or NA polypeptide (or peptide) or HA and/or NA RNA (e.g., anantisense RNA or ribozyme) into a population of target cells in vitro,ex vivo or in vivo. Typically, the polynucleotide encoding thepolypeptide (or peptide), or RNA, of interest is operably linked toappropriate regulatory sequences, e.g., as described herein. Optionally,more than one heterologous coding sequence is incorporated into a singlevector or virus. For example, in addition to a polynucleotide encoding atherapeutically or prophylactically active HA and/or NA polypeptide orRNA, the vector can also include additional therapeutic or prophylacticpolypeptides, e.g., antigens, co-stimulatory molecules, cytokines,antibodies, etc., and/or markers, and the like.

Although vaccination of an individual with an attenuated influenza virusof a particular strain of a particular subgroup can inducecross-protection against influenza virus of different strains and/orsubgroups, cross-protection can be enhanced, if desired, by vaccinatingthe individual with attenuated influenza virus from at least two, atleast three, or at least four influenza virus strains or substrains,e.g., at least two of which may represent a different subgroup. Forexample, vaccinating an individual with at least four strains orsubstrains of attenuated influenza virus may include vaccinating theindividual with at least two strains or substrains of influenza A virusand at least two strains or substrains of influenza B virus. Vaccinatingthe individual with the at least four strains or substrains ofattenuated influenza virus may include vaccinating the individual withat least three strains or substrains of influenza A virus and at leastone strain or substrain of influenza B virus. The vaccination of theindividual with at least four influenza virus strains or substrains mayrequire administration of a single tetravalent vaccine which comprisesall of the at least four attenuated influenza virus strains orsubstrains. The vaccination may alternatively require administration ofmultiple vaccines, each of which comprises one, two, or three of theattenuated influenza virus strains or substrains. Additionally, vaccinecombinations can optionally include mixes of pandemic vaccines andnon-pandemic strains. Vaccine mixtures (or multiple vaccinations) cancomprise components from human strains and/or non-human influenzastrains (e.g., avian and human, etc.). Similarly, the attenuatedinfluenza virus vaccines of this invention can optionally be combinedwith vaccines that induce protective immune responses against otherinfectious agents.

Polynucleotides of the Invention

Probes

The HA and NA polynucleotides of the invention, e.g., as shown in thesequences herein such as SEQ ID NO:1 through SEQ ID NO:48, and fragmentsthereof, are optionally used in a number of different capacitiesalternative to, or in addition to, the vaccines described above. Otherexemplary uses are described herein for illustrative purpose and not aslimitations on the actual range of uses, etc. Different methods ofconstruction, purification, and characterization of the nucleotidesequences of the invention are also described herein.

In some embodiments, nucleic acids including one or more polynucleotidesequence of the invention are favorably used as probes for the detectionof corresponding or related nucleic acids in a variety of contexts, suchas in nucleic hybridization experiments, e.g., to find and/orcharacterize homologous influenza variants (e.g., homologues tosequences herein, etc.) infecting other species or in differentinfluenza outbreaks, etc. The probes can be either DNA or RNA molecules,such as restriction fragments of genomic or cloned DNA, cDNAs, PCRamplification products, transcripts, and oligonucleotides, and can varyin length from oligonucleotides as short as about 10 nucleotides inlength to full length sequences or cDNAs in excess of 1 kb or more. Forexample, in some embodiments, a probe of the invention includes apolynucleotide sequence or subsequence selected, e.g., from among SEQ IDNO:1-SEQ ID NO:48, or sequences complementary thereto. Alternatively,polynucleotide sequences that are variants of one of theabove-designated sequences are used as probes. Most typically, suchvariants include one or a few conservative nucleotide variations. Forexample, pairs (or sets) of oligonucleotides can be selected, in whichthe two (or more) polynucleotide sequences are conservative variationsof each other, wherein one polynucleotide sequence correspondsidentically to a first variant or and the other(s) correspondsidentically to additional variants. Such pairs of oligonucleotide probesare particularly useful, e.g., for specific hybridization experiments todetect polymorphic nucleotides or to, e.g., detect homologous influenzaHA and NA variants, e.g., homologous to the current HA and NA sequences,infecting other species or present in different (e.g., either temporallyand/or geographically different) influenza outbreaks. In otherapplications, probes are selected that are more divergent, that isprobes that are at least about 91% (or about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, about 98.5%, about 98.7%,about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about99.5%, or about 99.6% or more about 99.7%, about 99.8%, about 99.9% ormore) identical are selected.

The probes of the invention, e.g., as exemplified by sequences derivedfrom the sequences herein, can also be used to identify additionaluseful polynucleotide sequences according to procedures routine in theart. In one set of embodiments, one or more probes, as described above,are utilized to screen libraries of expression products or chromosomalsegments (e.g., expression libraries or genomic libraries) to identifyclones that include sequences identical to, or with significant sequencesimilarity to, e.g., one or more probe of, e.g., SEQ ID NO:1-SEQ IDNO:48, i.e., variants, homologues, etc. It will be understood that inaddition to such physical methods as library screening, computerassisted bioinformatic approaches, e.g., BLAST and other sequencehomology search algorithms, and the like, can also be used foridentifying related polynucleotide sequences. Polynucleotide sequencesidentified in this manner are also a feature of the invention.

Oligonucleotide probes are optionally produced via a variety of methodswell known to those skilled in the art. Most typically, they areproduced by well known synthetic methods, such as the solid phasephosphoramidite triester method described by Beaucage and Caruthers(1981) Tetrahedron Letts 22(20):1859-1862, e.g., using an automatedsynthesizer, or as described in Needham-Van Devanter et al. (1984) NuclAcids Res, 12:6159-6168. Oligonucleotides can also be custom made andordered from a variety of commercial sources known to persons of skill.Purification of oligonucleotides, where necessary, is typicallyperformed by either native acrylamide gel electrophoresis or byanion-exchange HPLC as described in Pearson and Regnier (1983) J Chrom255:137-149. The sequence of the synthetic oligonucleotides can beverified using the chemical degradation method of Maxam and Gilbert(1980) in Grossman and Moldave (eds.) Academic Press, New York, Methodsin Enzymology 65:499-560. Custom oligos can also easily be ordered froma variety of commercial sources known to persons of skill.

In other circumstances, e.g., relating to attributes of cells ororganisms expressing the polynucleotides and polypeptides of theinvention (e.g., those harboring virus comprising the sequences of theinvention), probes that are polypeptides, peptides or antibodies arefavorably utilized. For example, isolated or recombinant polypeptides,polypeptide fragments and peptides derived from any of the amino acidsequences of the invention and/or encoded by polynucleotide sequences ofthe invention, e.g., selected from SEQ ID NO:1 through SEQ ID NO:48, arefavorably used to identify and isolate antibodies, e.g., from phagedisplay libraries, combinatorial libraries, polyclonal sera, and thelike.

Antibodies specific for any a polypeptide sequence or subsequence, e.g.,of SEQ ID NO:49 through SEQ ID NO:96, and/or encoded by polynucleotidesequences of the invention, e.g., selected from SEQ ID NO:1 through SEQID NO:48, are likewise valuable as probes for evaluating expressionproducts, e.g., from cells or tissues. In addition, antibodies areparticularly suitable for evaluating expression of proteins comprisingamino acid subsequences, e.g., of those given herein, or encoded bypolynucleotides sequences of the invention, e.g., selected from thoseshown herein, in situ, in a tissue array, in a cell, tissue or organism,e.g., an organism infected by an unidentified influenza virus or thelike. Antibodies can be directly labeled with a detectable reagent, ordetected indirectly by labeling of a secondary antibody specific for theheavy chain constant region (i.e., isotype) of the specific antibody.Antibodies against specific amino acids sequences herein (e.g., SEQ IDNOs: 49-96) are also useful in determining whether other influenzaviruses are within the same strain as the current sequences (e.g.,through an HI assay, etc.). Additional details regarding production ofspecific antibodies are provided below.

Diagnostic Assays

The nucleic acid sequences of the present invention can be used indiagnostic assays to detect influenza (and/or hemagglutinin and/orneuraminidase) in a sample, to detect hemagglutinin-like and/orneuraminidase-like sequences, and to detect strain differences inclinical isolates of influenza using either chemically synthesized orrecombinant polynucleotide fragments, e.g., selected from the sequencesherein. For example, fragments of the hemagglutinin and/or neuraminidasesequences comprising at least between 10 and 20 nucleotides can be usedas primers to amplify nucleic acids using polymerase chain reaction(PCR) methods well known in the art (e.g., reverse transcription-PCR)and as probes in nucleic acid hybridization assays to detect targetgenetic material such as influenza RNA in clinical specimens.

The probes of the invention, e.g., as exemplified by unique subsequencesselected from, e.g., SEQ ID NO:1 through SEQ ID NO:48, can also be usedto identify additional useful polynucleotide sequences (such as tocharacterize additional strains of influenza) according to proceduresroutine in the art. In one set of preferred embodiments, one or moreprobes, as described above, are utilized to screen libraries ofexpression products or cloned viral nucleic acids (i.e., expressionlibraries or genomic libraries) to identify clones that includesequences identical to, or with significant sequence identity to thesequences herein. In turn, each of these identified sequences can beused to make probes, including pairs or sets of variant probes asdescribed above. It will be understood that in addition to such physicalmethods as library screening, computer assisted bioinformaticapproaches, e.g., BLAST and other sequence homology search algorithms,and the like, can also be used for identifying related polynucleotidesequences.

The probes of the invention are particularly useful for detecting thepresence and for determining the identity of influenza nucleic acids incells, tissues or other biological samples (e.g., a nasal wash orbronchial lavage). For example, the probes of the invention arefavorably utilized to determine whether a biological sample, such as asubject (e.g., a human subject) or model system (such as a cultured cellsample) has been exposed to, or become infected with influenza, orparticular strain(s) of influenza. Detection of hybridization of theselected probe to nucleic acids originating in (e.g., isolated from) thebiological sample or model system is indicative of exposure to orinfection with the virus (or a related virus) from which the probepolynucleotide is selected.

It will be appreciated that probe design is influenced by the intendedapplication. For example, where several allele-specific probe-targetinteractions are to be detected in a single assay, e.g., on a single DNAchip, it is desirable to have similar melting temperatures for all ofthe probes. Accordingly, the lengths of the probes are adjusted so thatthe melting temperatures for all of the probes on the array are closelysimilar (it will be appreciated that different lengths for differentprobes may be needed to achieve a particular T_(m) where differentprobes have different GC contents). Although melting temperature is aprimary consideration in probe design, other factors are optionally usedto further adjust probe construction, such as selecting against primerself-complementarity and the like.

Vectors, Promoters and Expression Systems

The present invention includes recombinant constructs incorporating oneor more of the nucleic acid sequences described herein. Such constructsoptionally include a vector, for example, a plasmid, a cosmid, a phage,a virus, a bacterial artificial chromosome (BAC), a yeast artificialchromosome (YAC), etc., into which one or more of the polynucleotidesequences of the invention, e.g., comprising any of SEQ ID NO:1 throughSEQ ID NO:48, or a subsequence thereof etc., has been inserted, in aforward or reverse orientation. For example, the inserted nucleic acidcan include a viral chromosomal sequence or cDNA including all or partof at least one of the polynucleotide sequences of the invention. In oneembodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available.

The polynucleotides of the present invention can be included in any oneof a variety of vectors suitable for generating sense or antisense RNA,and optionally, polypeptide (or peptide) expression products (e.g., ahemagglutinin and/or neuraminidase molecule of the invention, orhemagglutinin or neuraminidase fragments). Such vectors includechromosomal, nonchromosomal and synthetic DNA sequences, e.g.,derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeastplasmids; vectors derived from combinations of plasmids and phage DNA,viral DNA such as vaccinia, adenovirus, fowl pox virus, pseudorabies,adenovirus, adeno-associated virus, retroviruses and many others (e.g.,pCDL). Any vector that is capable of introducing genetic material into acell, and, if replication is desired, which is replicable in therelevant host can be used.

In an expression vector, the HA and/or NA polynucleotide sequence ofinterest is physically arranged in proximity and orientation to anappropriate transcription control sequence (e.g., promoter, andoptionally, one or more enhancers) to direct mRNA synthesis. That is,the polynucleotide sequence of interest is operably linked to anappropriate transcription control sequence. Examples of such promotersinclude: LTR or SV40 promoter, E. coli lac or trp promoter, phage lambdaP_(L) promoter, and other promoters known to control expression of genesin prokaryotic or eukaryotic cells or their viruses.

A variety of promoters are suitable for use in expression vectors forregulating transcription of influenza virus genome segments. In certainembodiments, the cytomegalovirus (CMV) DNA dependent RNA Polymerase II(Pol II) promoter is utilized. If desired, e.g., for regulatingconditional expression, other promoters can be substituted which induceRNA transcription under the specified conditions, or in the specifiedtissues or cells. Numerous viral and mammalian, e.g., human promotersare available, or can be isolated according to the specific applicationcontemplated. For example, alternative promoters obtained from thegenomes of animal and human viruses include such promoters as theadenovirus (such as Adenovirus 2), papilloma virus, hepatitis-B virus,polyoma virus, and Simian Virus 40 (SV40), and various retroviralpromoters. Mammalian promoters include, among many others, the actinpromoter, immunoglobulin promoters, heat-shock promoters, and the like.

Various embodiments of the current invention can comprise a number ofdifferent vector constructions. Such constructions are typically andpreferably used in plasmid rescue systems to create viruses for use invaccines (e.g., in live attenuated vaccines, in killed or inactivatedvaccines, etc.). Thus, the invention includes recombinant DNA moleculeshaving a transcription control element that binds a DNA-directed RNApolymerase that is operatively linked to a DNA sequence that encodes anRNA molecule, wherein the RNA molecule comprises a binding site specificfor an RNA-directed RNA polymerase of a negative strand RNA virus,operatively linked to an RNA sequence comprising the reverse complementof a mRNA coding sequence of a negative strand RNA virus. Also, theinvention includes a recombinant DNA molecule that, upon transcriptionyields an RNA template that contains an RNA sequence comprising thereverse complement of an mRNA coding sequence of a negative strand RNAvirus, and vRNA terminal sequences. The invention also includes arecombinant DNA molecule that upon transcription yields a replicable RNAtemplate comprising the reverse complement of an mRNA coding sequence ofa negative strand RNA virus. Such above recombinant DNA moleculestypically involve wherein the negative strand RNA virus is influenza(e.g., influenza A or B, etc.). Also, the RNA molecule in suchembodiments is typically an influenza genome segment and the RNAtemplate is typically an influenza genome segment. The recombinant DNAmolecules typically comprise wherein the RNA template is replicable,wherein the negative strand RNA virus is influenza, and wherein the RNAtemplate is an influenza genome segment. Thus, the nucleic acidsinfluenza segments typically comprise HA and/or NA genes (thecorresponding nucleic acid of which is, e.g., in FIG. 1, or withinsimilar strains of the strains having the nucleic acids in, e.g., FIG.1.

The invention also includes methods of preparing an RNA moleculecomprising transcribing a recombinant DNA molecule with a DNA-directedRNA polymerase, wherein the DNA molecule comprises a transcriptioncontrol element that binds a DNA-directed RNA polymerase that isoperatively linked to a DNA sequence that encodes an RNA molecule,wherein the RNA molecule comprises a binding site specific for anRNA-directed RNA polymerase of a negative strand RNA virus, operativelylinked to an RNA sequence comprising the reverse complement of an mRNAcoding sequence of a negative strand RNA virus. The invention alsoincludes a method of preparing an RNA molecule comprising transcribing arecombinant DNA molecule with a DNA-directed RNA polymerase, wherein therecombinant DNA molecule yields upon transcription an RNA molecule thatcontains an RNA sequence comprising the reverse complement of an mRNAcoding sequence of a negative strand RNA virus, and vRNA terminalsequences. Furthermore, the invention includes a method of preparing anRNA molecule comprising transcribing a recombinant DNA molecule with aDNA-directed RNA polymerase, wherein the recombinant DNA molecule yieldsupon transcription a replicable RNA molecule comprising the reversecomplement of an mRNA coding sequence of a negative strand RNA virus.Such methods typically comprise wherein the negative strand RNA virus isinfluenza, and wherein the RNA molecule is an influenza genome segment.Such methods preferably include wherein the DNA-directed RNA polymeraseis pol I, pol II, T7 polymerase, T3 polymerase, or Sp6 polymerase. Thus,again, the influenza nucleic acid segments typically comprise HA and/orNA genes as described throughout.

Other methods within the invention include methods of constructing a DNAmolecule comprising a transcription control element that binds aDNA-directed RNA polymerase that is operatively linked to a DNA sequencethat encodes an RNA molecule, wherein the RNA molecule comprises abinding site specific for an RNA-directed RNA polymerase of an influenzavirus, operatively linked to an RNA sequence comprising the reversecomplement of an mRNA coding sequence of an influenza virus, wherein theDNA sequence comprises a nucleic acid corresponding to one or more ofSEQ ID NO:1-48 or a fragment thereof or of one or more nucleic acidsequence of a similar strain (e.g., a strain similar to such strainshaving the sequences found in the sequences of FIG. 1, etc.). Also, theinvention includes a method of constructing a DNA molecule comprising aDNA sequence that upon transcription yields an RNA template thatcontains an RNA sequence comprising the reverse complement of an mRNAcoding sequence of an influenza virus, and vRNA terminal sequences,wherein the DNA sequence comprises a nucleic acid corresponding to oneor more of SEQ ID NO:1-48 or a fragment thereof, or of one or morenucleic acid of a similar strain (e.g., a strain similar to such strainsthat have the sequences found in FIG. 1, etc.). Such methods alsoinclude wherein the RNA template is replicable. Other methods of theinvention include those of constructing a DNA molecule comprising a DNAsequence that upon transcription yields a replicable RNA templatecomprising the reverse complement of an mRNA coding sequence of aninfluenza virus. These methods of the invention typically includewherein the RNA molecule is an influenza genome segment, wherein theDNA-directed RNA polymerase is pol I, pol II, T7 polymerase, T3polymerase, or Sp6 polymerase.

Transcription is optionally increased by including an enhancer sequence.Enhancers are typically short, e.g., 10-500 bp, cis-acting DNA elementsthat act in concert with a promoter to increase transcription. Manyenhancer sequences have been isolated from mammalian genes (hemoglobin,elastase, albumin, alpha-fetoprotein, and insulin), and eukaryotic cellviruses. The enhancer can be spliced into the vector at a position 5′ or3′ to the heterologous coding sequence, but is typically inserted at asite 5′ to the promoter. Typically, the promoter, and if desired,additional transcription enhancing sequences are chosen to optimizeexpression in the host cell type into which the heterologous DNA is tobe introduced (Scharf et al. (1994) Heat stress promoters andtranscription factors Results Probl Cell Differ 20:125-62; Kriegler etal. (1990) Assembly of enhancers, promoters, and splice signals tocontrol expression of transferred genes Methods in Enzymol 185: 512-27).Optionally, the amplicon can also contain a ribosome binding site or aninternal ribosome entry site (IRES) for translation initiation.

The vectors of the invention also favorably include sequences necessaryfor the termination of transcription and for stabilizing the mRNA, suchas a polyadenylation site or a terminator sequence. Such sequences arecommonly available from the 5′ and, occasionally 3′, untranslatedregions of eukaryotic or viral DNAs or cDNAs. In one embodiment, theSV40 polyadenylation signal sequences can provide a bi-directionalpolyadenylation site that insulates transcription of (+) strand mRNAmolecules from the Poll promoter initiating replication of the (−)strand viral genome.

In addition, as described above, the expression vectors optionallyinclude one or more selectable marker genes to provide a phenotypictrait for selection of transformed host cells, in addition to genespreviously listed, markers such as dihydrofolate reductase or neomycinresistance are suitable for selection in eukaryotic cell culture.

The vector containing the appropriate nucleic acid sequence as describedabove, as well as an appropriate promoter or control sequence, can beemployed to transform a host cell permitting expression of the protein.While the vectors of the invention can be replicated in bacterial cells,most frequently it will be desirable to introduce them into mammaliancells, e.g., Vero cells, BHK cells, MDCK cell, 293 cells, COS cells, orthe like, for the purpose of expression.

As described elsewhere, the HA and NA sequences herein, in variousembodiments, can be comprised within plasmids involved in plasmid-rescuereassortment. See, e.g., U.S. Application No. 60/420,708, filed Oct. 23,2002, U.S. application Ser. No. 10/423,828, filed Apr. 25, 2003, andU.S. Application No. 60/574,117, filed May 24, 2004, all entitled“Multi-Plasmid System for the Production of Influenza Virus”; Hoffmann,E., 2000, PNAS, 97(11):6108-6113; U.S. Published Patent Application No.20020164770 to Hoffmann; and U.S. Pat. No. 6,544,785 issued Apr. 8, 2003to Palese, et al. The reassortants produced can include the HA and NAgenes arranged with the 6 other influenza genes from the A/AnnArbor/6/60 donor strain, the B/Ann Arbor/1/66 donor strain (and/orderivatives and modifications thereof), the A/Puerto Rico/8/34 donorstrain, etc.

Additional Expression Elements

Most commonly, the genome segment encoding the influenza virus HA and/orNA protein includes any additional sequences necessary for itsexpression, including translation into a functional viral protein. Inother situations, a minigene, or other artificial construct encoding theviral proteins, e.g., an HA and/or NA protein, can be employed. Again,in such case, it is often desirable to include specific initiationsignals that aid in the efficient translation of the heterologous codingsequence. These signals can include, e.g., the ATG initiation codon andadjacent sequences. To insure translation of the entire insert, theinitiation codon is inserted in the correct reading frame relative tothe viral protein. Exogenous transcriptional elements and initiationcodons can be of various origins, both natural and synthetic. Theefficiency of expression can be enhanced by the inclusion of enhancersappropriate to the cell system in use.

If desired, polynucleotide sequences encoding additional expressedelements, such as signal sequences, secretion or localization sequences,and the like can be incorporated into the vector, usually, in-frame withthe polynucleotide sequence of interest, e.g., to target polypeptideexpression to a desired cellular compartment, membrane, or organelle, orto direct polypeptide secretion to the periplasmic space or into thecell culture media. Such sequences are known to those of skill, andinclude secretion leader peptides, organelle targeting sequences (e.g.,nuclear localization sequences, ER retention signals, mitochondrialtransit sequences), membrane localization/anchor sequences (e.g., stoptransfer sequences, GPI anchor sequences), and the like.

Where translation of a polypeptide encoded by a nucleic acid sequence ofthe invention is desired, additional translation specific initiationsignals can improve the efficiency of translation. These signals caninclude, e.g., an ATG initiation codon and adjacent sequences, an IRESregion, etc. In some cases, for example, full-length cDNA molecules orchromosomal segments including a coding sequence incorporating, e.g., apolynucleotide sequence of the invention (e.g., as in the sequencesherein), a translation initiation codon and associated sequence elementsare inserted into the appropriate expression vector simultaneously withthe polynucleotide sequence of interest. In such cases, additionaltranslational control signals frequently are not required. However, incases where only a polypeptide coding sequence, or a portion thereof, isinserted, exogenous translational control signals, including, e.g., anATG initiation codon is often provided for expression of the relevantsequence. The initiation codon is put in the correct reading frame toensure transcription of the polynucleotide sequence of interest.Exogenous transcriptional elements and initiation codons can be ofvarious origins, both natural and synthetic. The efficiency ofexpression can be enhanced by the inclusion of enhancers appropriate tothe cell system in use (see, e.g., Scharf D. et al. (1994) Results ProblCell Differ 20:125-62; Bittner et al. (1987) Methods in Enzymol153:516-544).

Production of Recombinant Virus

Negative strand RNA viruses can be genetically engineered and recoveredusing a recombinant reverse genetics approach (U.S. Pat. No. 5,166,057to Palese et al.). Such method was originally applied to engineerinfluenza viral genomes (Luytjes et al. (1989) Cell 59:1107-1113; Enamiet al. (1990) Proc. Natl. Acad. Sci. USA 92:11563-11567), and has beensuccessfully applied to a wide variety of segmented and nonsegmentednegative strand RNA viruses, e.g., rabies (Schnell et al. (1994) EMBO J.13: 4195-4203); VSV (Lawson et al. (1995) Proc. Natl. Acad. Sci. USA 92:4477-4481); measles virus (Radecke et al. (1995) EMBO J. 14:5773-5784);rinderpest virus (Baron & Barrett (1997) J. Virol. 71: 1265-1271); humanparainfluenza virus (Hoffman & Banerjee (1997) J. Virol. 71: 3272-3277;Dubin et al. (1997) Virology 235:323-332); SV5 (He et al. (1997)Virology 237:249-260); canine distemper virus (Gassen et al. (2000) J.Virol. 74:10737-44); and Sendai virus (Park et al. (1991) Proc. Natl.Acad. Sci. USA 88: 5537-5541; Kato et al. (1996) Genes to Cells1:569-579). Those of skill in the art will be familiar with these andsimilar techniques to produce influenza virus comprising the HA and NAsequences of the invention. Recombinant influenza viruses producedaccording to such methods are also a feature of the invention, as arerecombinant influenza virus comprising one or more nucleic acids and/orpolypeptides of the invention. Of course, as will be appreciated bythose of skill in the art, influenza viruses in general (and those ofthe invention as well) are negative stranded RNA viruses. Thus, when thepresent invention describes influenza viruses as comprising, e.g., thesequences of FIG. 1, etc., it is to be understood to typically mean thecorresponding negative stranded RNA version of the sequences. Thenucleotide sequences in FIG. 1 comprise DNA versions (e.g., coding plussense, etc.) of the genes (along with some untranslated regions in thenucleotide sequences). Those of skill in the art can easily convertbetween RNA and DNA sequences (e.g., changing U to T, etc.), and betweencomplementary nucleotide sequences (whether RNA or DNA), etc. Thus, forexample, those of skill in the art can easily convert from a nucleotidesequence (e.g., one given in FIG. 1 such as SEQ ID NO:1) to thecorresponding amino acid sequence or to a corresponding complementarysequence (whether DNA or RNA), etc. Also, as will be evident, when suchHA and/or NA sequences are described within DNA vectors, e.g., plasmids,etc., then the corresponding DNA version of the sequences are typicallyto be understood. Again, nucleic acids of the invention include theexplicit sequences in the sequence listings herein, as well as thecomplements of such sequences (both RNA and DNA), the double strandedform of the sequences in the sequence listings, the corresponding RNAforms of the sequences in the sequence listings (either as the RNAcomplement to the explicit sequence in the sequence listing or as theRNA version of the sequence in the sequence listing, e.g., of the samesense, but comprised of RNA, with U in place of T, etc.). Thus,depending on context herein, the nucleic acid sequences of the inventioncan comprise the RNA versions of SEQ ID NO: 1-48 (either in positivestranded sense or in negative stranded sense).

Cell Culture and Expression Hosts

The present invention also relates to host cells that are introduced(transduced, transformed or transfected) with vectors of the invention,and the production of polypeptides of the invention by recombinanttechniques. Host cells are genetically engineered (i.e., transduced,transformed or transfected) with a vector, such as an expression vector,of this invention. As described above, the vector can be in the form ofa plasmid, a viral particle, a phage, etc. Examples of appropriateexpression hosts include: bacterial cells, such as E. coli,Streptomyces, and Salmonella typhimurium; fungal cells, such asSaccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; orinsect cells such as Drosophila and Spodoptera frugiperda.

Most commonly, mammalian cells are used to culture the HA and NAmolecules of the invention. Suitable host cells for the replication ofinfluenza virus (e.g., with the HA and/or NA sequences herein) include,e.g., Vero cells, BHK cells, MDCK cells, 293 cells and COS cells,including 293T cells, COS7 cells or the like. Commonly, co-culturesincluding two of the above cell lines, e.g., MDCK cells and either 293Tor COS cells are employed at a ratio, e.g., of 1:1, to improvereplication efficiency. Typically, cells are cultured in a standardcommercial culture medium, such as Dulbecco's modified Eagle's mediumsupplemented with serum (e.g., 10% fetal bovine serum), or in serum freemedium, under controlled humidity and CO₂ concentration suitable formaintaining neutral buffered pH (e.g., at pH between 7.0 and 7.2).Optionally, the medium contains antibiotics to prevent bacterial growth,e.g., penicillin, streptomycin, etc., and/or additional nutrients, suchas L-glutamine, sodium pyruvate, non-essential amino acids, additionalsupplements to promote favorable growth characteristics, e.g., trypsin,β-mercaptoethanol, and the like.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants, or amplifying the inserted polynucleotide sequences,e.g., through production of viruses. The culture conditions, such astemperature, pH and the like, are typically those previously used withthe particular host cell selected for expression, and will be apparentto those skilled in the art and in the references cited herein,including, e.g., Freshney (1994) Culture of Animal Cells, a Manual ofBasic Technique, 3^(rd) edition, Wiley-Liss, New York and the referencescited therein. Other helpful references include, e.g., Paul (1975) Celland Tissue Culture, 5^(th) ed., Livingston, Edinburgh; Adams (1980)Laboratory Techniques in Biochemistry and Molecular Biology-Cell Culturefor Biochemists, Work and Burdon (eds.) Elsevier, Amsterdam. Additionaldetails regarding tissue culture procedures of particular interest inthe production of influenza virus in vitro include, e.g., Merten et al.(1996) Production of influenza virus in cell cultures for vaccinepreparation. in Cohen and Shafferman (eds.) Novel Strategies in Designand Production of Vaccines, which is incorporated herein in its entiretyfor all purposes. Additionally, variations in such procedures adapted tothe present invention are readily determined through routineexperimentation and will be familiar to those skilled in the art.

Cells for production of influenza virus (e.g., having the HA and/or NAsequences of the invention) can be cultured in serum-containing or serumfree medium. In some cases, e.g., for the preparation of purifiedviruses, it is typically desirable to grow the host cells in serum freeconditions. Cells can be cultured in small scale, e.g., less than 25 mlmedium, culture tubes or flasks or in large flasks with agitation, inrotator bottles, or on microcarrier beads (e.g., DEAE-Dextranmicrocarrier beads, such as Dormacell, Pfeifer & Langen; Superbead, FlowLaboratories; styrene copolymer-tri-methylamine beads, such as Hillex,SoloHill, Ann Arbor) in flasks, bottles or reactor cultures.Microcarrier beads are small spheres (in the range of 100-200 microns indiameter) that provide a large surface area for adherent cell growth pervolume of cell culture. For example a single liter of medium can includemore than 20 million microcarrier beads providing greater than 8000square centimeters of growth surface. For commercial production ofviruses, e.g., for vaccine production, it is often desirable to culturethe cells in a bioreactor or fermenter. Bioreactors are available involumes from under 1 liter to in excess of 100 liters, e.g., Cyto3Bioreactor (Osmonics, Minnetonka, Minn.); NBS bioreactors (New BrunswickScientific, Edison, N.J.); laboratory and commercial scale bioreactorsfrom B. Braun Biotech International (B. Braun Biotech, Melsungen,Germany).

Regardless of the culture volume, in many desired aspects of the currentinvention, it is important that the cultures be maintained at anappropriate temperature, to insure efficient recovery of recombinantand/or reassortant influenza virus using temperature dependent multiplasmid systems (see, e.g., U.S. Application No. 60/420,708, filed Oct.23, 2002, U.S. application Ser. No. 10/423,828, filed Apr. 25, 2003, andU.S. Application No. 60/574,117, filed May 24, 2004, all entitled“Multi-Plasmid System for the Production of Influenza Virus”), heatingof virus solutions for filtration, etc. Typically, a regulator, e.g., athermostat, or other device for sensing and maintaining the temperatureof the cell culture system and/or other solution, is employed to insurethat the temperature is at the correct level during the appropriateperiod (e.g., virus replication, etc.).

In some embodiments herein (e.g., wherein reassorted viruses are to beproduced from segments on vectors) vectors comprising influenza genomesegments are introduced (e.g., transfected) into host cells according tomethods well known in the art for introducing heterologous nucleic acidsinto eukaryotic cells, including, e.g., calcium phosphateco-precipitation, electroporation, microinjection, lipofection, andtransfection employing polyamine transfection reagents. For example,vectors, e.g., plasmids, can be transfected into host cells, such as COScells, 293T cells or combinations of COS or 293T cells and MDCK cells,using the polyamine transfection reagent TranslT-LT1 (Mirus) accordingto the manufacturer's instructions in order to produce reassortedviruses, etc. Thus, in one example, approximately 1 μg of each vector isintroduced into a population of host cells with approximately 2 μl ofTransIT-LT1 diluted in 160 μl medium, preferably serum-free medium, in atotal volume of 200 μl. The DNA:transfection reagent mixtures areincubated at room temperature for 45 minutes followed by addition of 800μl of medium. The transfection mixture is added to the host cells, andthe cells are cultured as described via other methods well known tothose skilled in the art. Accordingly, for the production of recombinantor reassortant viruses in cell culture, vectors incorporating each ofthe 8 genome segments, (PB2, PB1, PA, NP, M, NS, HA and NA, e.g., of theinvention) are mixed with approximately 20 μl TranslT-LT1 andtransfected into host cells. Optionally, serum-containing medium isreplaced prior to transfection with serum-free medium, e.g., Opti-MEM I,and incubated for 4-6 hours.

Alternatively, electroporation can be employed to introduce such vectorsincorporating influenza genome segments into host cells. For example,plasmid vectors incorporating an influenza A or influenza B virus arefavorably introduced into Vero cells using electroporation according tothe following procedure. In brief, approximately 5×10⁶ Vero cells, e.g.,grown in Modified Eagle's Medium (MEM) supplemented with 10% FetalBovine Serum (FBS) are resuspended in 0.4 ml OptiMEM and placed in anelectroporation cuvette. Twenty micrograms of DNA in a volume of up to25 μl is added to the cells in the cuvette, which is then mixed gentlyby tapping. Electroporation is performed according to the manufacturer'sinstructions (e.g., BioRad Gene Pulser II with Capacitance Extender Plusconnected) at 300 volts, 950 microFarads with a time constant of between28-33 msec. The cells are remixed by gently tapping and approximately1-2 minutes following electroporation 0.7 ml MEM with 10% FBS is addeddirectly to the cuvette. The cells are then transferred to two wells ofa standard 6 well tissue culture dish containing 2 ml MEM, 10% FBS. Thecuvette is washed to recover any remaining cells and the wash suspensionis divided between the two wells. Final volume is approximately 3.5 mL.The cells are then incubated under conditions permissive for viralgrowth, e.g., at approximately 33° C. for cold adapted strains.

In mammalian host cells, a number of expression systems, such asviral-based systems, can be utilized. In cases where an adenovirus isused as an expression vector, a coding sequence is optionally ligatedinto an adenovirus transcription/translation complex consisting of thelate promoter and tripartite leader sequence. Insertion in anonessential E1 or E3 region of the viral genome will result in a viablevirus capable of expressing the polypeptides of interest in infectedhost cells (Logan and Shenk (1984) Proc Natl Acad Sci 81:3655-3659). Inaddition, transcription enhancers, such as the rous sarcoma virus (RSV)enhancer, can be used to increase expression in mammalian host cells.

A host cell strain is optionally chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed proteinin the desired fashion. Such modifications of the protein include, butare not limited to, acetylation, carboxylation, glycosylation,phosphorylation, lipidation and acylation. Post-translationalprocessing, which cleaves a precursor form into a mature form, of theprotein is sometimes important for correct insertion, folding and/orfunction. Additionally proper location within a host cell (e.g., on thecell surface) is also important. Different host cells such as COS, CHO,BHK, MDCK, 293, 293T, COS7, etc. have specific cellular machinery andcharacteristic mechanisms for such post-translational activities and canbe chosen to ensure the correct modification and processing of thecurrent introduced, foreign protein.

For long-term, high-yield production of recombinant proteins encoded by,or having subsequences encoded by, the polynucleotides of the invention,stable expression systems are optionally used. For example, cell lines,stably expressing a polypeptide of the invention, are transfected usingexpression vectors that contain viral origins of replication orendogenous expression elements and a selectable marker gene. Forexample, following the introduction of the vector, cells are allowed togrow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells that successfully express the introduced sequences. Thus,resistant clumps of stably transformed cells, e.g., derived from singlecell type, can be proliferated using tissue culture techniquesappropriate to the cell type.

Host cells transformed with a nucleotide sequence encoding a polypeptideof the invention are optionally cultured under conditions suitable forthe expression and recovery of the encoded protein from cell culture.The cells expressing said protein can be sorted, isolated and/orpurified. The protein or fragment thereof produced by a recombinant cellcan be secreted, membrane-bound, or retained intracellularly, dependingon the sequence (e.g., depending upon fusion proteins encoding amembrane retention signal or the like) and/or the vector used.

Expression products corresponding to the nucleic acids of the inventioncan also be produced in non-animal cells such as plants, yeast, fungi,bacteria and the like. In addition to Sambrook, Berger and Ausubel, allinfra, details regarding cell culture can be found in Payne et al.(1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds.) (1995) PlantCell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg N.Y.) and Atlas and Parks (eds.) TheHandbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.

In bacterial systems, a number of expression vectors can be selecteddepending upon the use intended for the expressed product. For example,when large quantities of a polypeptide or fragments thereof are neededfor the production of antibodies, vectors that direct high-levelexpression of fusion proteins that are readily purified are favorablyemployed. Such vectors include, but are not limited to, multifunctionalE. coli cloning and expression vectors such as BLUESCRIPT (Stratagene),in which the coding sequence of interest, e.g., sequences comprisingthose found herein, etc., can be ligated into the vector in-frame withsequences for the amino-terminal translation initiating methionine andthe subsequent 7 residues of beta-galactosidase producing acatalytically active beta galactosidase fusion protein; pIN vectors (VanHeeke & Schuster (1989) J Biol Chem 264:5503-5509); pET vectors(Novagen, Madison Wis.); and the like. Similarly, in the yeastSaccharomyces cerevisiae a number of vectors containing constitutive orinducible promoters such as alpha factor, alcohol oxidase and PGH can beused for production of the desired expression products. For reviews, seeAusubel, infra, and Grant et al., (1987); Methods in Enzymology153:516-544.

Nucleic Acid Hybridization

Comparative hybridization can be used to identify nucleic acids of theinvention, including conservative variations of nucleic acids of theinvention. This comparative hybridization method is a preferred methodof distinguishing nucleic acids of the invention. In addition, targetnucleic acids which hybridize to the nucleic acids represented by, e.g.,SEQ ID NO:1 through SEQ ID NO:48 under high, ultra-high andultra-ultra-high stringency conditions are features of the invention.Examples of such nucleic acids include those with one or a few silent orconservative nucleic acid substitutions as compared to a given nucleicacid sequence.

A test target nucleic acid is said to specifically hybridize to a probenucleic acid when it hybridizes at least one-half as well to the probeas to the perfectly matched complementary target, i.e., with a signal tonoise ratio at least one-half as high as hybridization of the probe tothe target under conditions in which the perfectly matched probe bindsto the perfectly matched complementary target with a signal to noiseratio that is at least about 5×-10× as high as that observed forhybridization to any of the unmatched target nucleic acids.

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well-characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. Numerous protocols for nucleic acidhybridization are well known in the art. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology—Hybridization withNucleic Acid Probes part I chapter 2, “Overview of principles ofhybridization and the strategy of nucleic acid probe assays,” (Elsevier,N.Y.), as well as in Ausubel, Sambrook, and Berger and Kimmel, allbelow. Hames and Higgins (1995) Gene Probes 1 IRL Press at OxfordUniversity Press, Oxford, England, (Hames and Higgins 1) and Hames andHiggins (1995) Gene Probes 2 IRL Press at Oxford University Press,Oxford, England (Hames and Higgins 2) provide details on the synthesis,labeling, detection and quantification of DNA and RNA, includingoligonucleotides.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or northern blot is 50% formalin with1 mg of heparin at 42° C., with the hybridization being carried outovernight. An example of stringent wash conditions comprises a 0.2×SSCwash at 65° C. for 15 minutes (see, Sambrook, infra for a description ofSSC buffer). Often the high stringency wash is preceded by a lowstringency wash to remove background probe signal. An example lowstringency wash is 2×SSC at 40° C. for 15 minutes. In general, a signalto noise ratio of 5× (or higher) than that observed for an unrelatedprobe in the particular hybridization assay indicates detection of aspecific hybridization.

After hybridization, unhybridized nucleic acids can be removed by aseries of washes, the stringency of which can be adjusted depending uponthe desired results. Low stringency washing conditions (e.g., usinghigher salt and lower temperature) increase sensitivity, but can producenonspecific hybridization signals and high background signals. Higherstringency conditions (e.g., using lower salt and higher temperaturethat is closer to the T_(m)) lower the background signal, typically withprimarily the specific signal remaining. See, also, Rapley, R. andWalker, J. M. eds., Molecular Biomethods Handbook (Humana Press, Inc.1998).

“Stringent hybridization wash conditions” in the context of nucleic acidhybridization experiments such as Southern and northern hybridizationsare sequence dependent, and are different under different environmentalparameters. An extensive guide to the hybridization of nucleic acids isfound in Tijssen (1993), supra, and in Hames and Higgins, 1 and 2.Stringent hybridization and wash conditions can easily be determinedempirically for any test nucleic acid. For example, in determininghighly stringent hybridization and wash conditions, the hybridizationand wash conditions are gradually increased (e.g., by increasingtemperature, decreasing salt concentration, increasing detergentconcentration and/or increasing the concentration of organic solventssuch as formalin in the hybridization or wash), until a selected set ofcriteria is met. For example, the hybridization and wash conditions aregradually increased until a probe binds to a perfectly matchedcomplementary target with a signal to noise ratio that is at least 5× ashigh as that observed for hybridization of the probe to an unmatchedtarget.

In general, a signal to noise ratio of at least 2× (or higher, e.g., atleast 5×, 10×, 20×, 50×, 100×, or more) than that observed for anunrelated probe in the particular hybridization assay indicatesdetection of a specific hybridization. Detection of at least stringenthybridization between two sequences in the context of the presentinvention indicates relatively strong structural similarity to, e.g.,the nucleic acids of the present invention provided in the sequencelistings herein.

“Very stringent” conditions are selected to be equal to the thermalmelting point (T_(m)) for a particular probe. The T_(m), is thetemperature (under defined ionic strength and pH) at which 50% of thetest sequence hybridizes to a perfectly matched probe. For the purposesof the present invention, generally, “highly stringent” hybridizationand wash conditions are selected to be about 5° C. lower than the T_(m)for the specific sequence at a defined ionic strength and pH (as notedbelow, highly stringent conditions can also be referred to incomparative terms). Target sequences that are closely related oridentical to the nucleotide sequence of interest (e.g., “probe”) can beidentified under stringent or highly stringent conditions. Lowerstringency conditions are appropriate for sequences that are lesscomplementary.

“Ultra high-stringency” hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10× ashigh as that observed for hybridization to any unmatched target nucleicacids. A target nucleic acid which hybridizes to a probe under suchconditions, with a signal to noise ratio of at least one-half that ofthe perfectly matched complementary target nucleic acid is said to bindto the probe under ultra-high stringency conditions.

In determining stringent or highly stringent hybridization (or even morestringent hybridization) and wash conditions, the hybridization and washconditions are gradually increased (e.g., by increasing temperature,decreasing salt concentration, increasing detergent concentration and/orincreasing the concentration of organic solvents, such as formamide, inthe hybridization or wash), until a selected set of criteria are met.For example, the hybridization and wash conditions are graduallyincreased until a probe comprising one or more polynucleotide sequencesof the invention, e.g., sequences or unique subsequences selected fromthose given herein and/or complementary polynucleotide sequences, bindsto a perfectly matched complementary target (again, a nucleic acidcomprising one or more nucleic acid sequences or subsequences selectedfrom those given herein and/or complementary polynucleotide sequencesthereof), with a signal to noise ratio that is at least 2× (andoptionally 5×, 10×, or 100× or more) as high as that observed forhybridization of the probe to an unmatched target (e.g., apolynucleotide sequence comprising one or more sequences or subsequencesselected from known influenza sequences present in public databases suchas GenBank at the time of filing, and/or complementary polynucleotidesequences thereof), as desired.

Using the polynucleotides of the invention, or subsequences thereof,novel target nucleic acids can be obtained; such target nucleic acidsare also a feature of the invention. For example, such target nucleicacids include sequences that hybridize under stringent conditions to aunique oligonucleotide probe corresponding to any of the polynucleotidesof the invention.

Similarly, even higher levels of stringency can be determined bygradually increasing the hybridization and/or wash conditions of therelevant hybridization assay. For example, those in which the stringencyof hybridization and wash conditions are increased until the signal tonoise ratio for binding of the probe to the perfectly matchedcomplementary target nucleic acid is at least 10×, 20×, 50×, 100×, or500× or more as high as that observed for hybridization to any unmatchedtarget nucleic acids. The particular signal will depend on the labelused in the relevant assay, e.g., a fluorescent label, a colorimetriclabel, a radioactive label, or the like. A target nucleic acid whichhybridizes to a probe under such conditions, with a signal to noiseratio of at least one-half that of the perfectly matched complementarytarget nucleic acid, is said to bind to the probe under ultra-ultra-highstringency conditions.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, e.g., when a copyof a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Cloning, Mutagenesis and Expression of Biomolecules of Interest

General texts which describe molecular biological techniques, which areapplicable to the present invention, such as cloning, mutation, cellculture and the like, include Berger and Kimmel, Guide to MolecularCloning Techniques, Methods in Enzymology volume 152 Academic Press,Inc., San Diego, Calif. (Berger); Sambrook et al., Molecular Cloning—ALaboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 2000 (“Sambrook”) and Current Protocols inMolecular Biology, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley& Sons, Inc., (supplemented through 2002) (“Ausubel”)). These textsdescribe mutagenesis, the use of vectors, promoters and many otherrelevant topics related to, e.g., the generation of HA and/or NAmolecules, etc.

Various types of mutagenesis are optionally used in the presentinvention, e.g., to produce and/or isolate, e.g., novel or newlyisolated HA and/or NA molecules and/or to further modify/mutate thepolypeptides (e.g., HA and NA molecules) of the invention. They includebut are not limited to site-directed, random point mutagenesis,homologous recombination (DNA shuffling), mutagenesis using uracilcontaining templates, oligonucleotide-directed mutagenesis,phosphorothioate-modified DNA mutagenesis, mutagenesis using gappedduplex DNA or the like. Additional suitable methods include pointmismatch repair, mutagenesis using repair-deficient host strains,restriction-selection and restriction-purification, deletionmutagenesis, mutagenesis by total gene synthesis, double-strand breakrepair, and the like. Mutagenesis, e.g., involving chimeric constructs,is also included in the present invention. In one embodiment,mutagenesis can be guided by known information of the naturallyoccurring molecule or altered or mutated naturally occurring molecule,e.g., sequence, sequence comparisons, physical properties, crystalstructure or the like.

The above texts and examples found herein describe these procedures aswell as the following publications (and references cited within):Sieber, et al., Nature Biotechnology, 19:456-460 (2001); Ling et al.,Approaches to DNA mutagenesis: an overview, Anal Biochem 254(2): 157-178(1997); Dale et al., Oligonucleotide-directed random mutagenesis usingthe phosphorothioate method, Methods Mol Biol 57:369-374 (1996); I. A.Lorimer, I. Pastan, Nucleic Acids Res 23, 3067-8 (1995); W. P. C.Stemmer, Nature 370, 389-91 (1994); Arnold, Protein engineering forunusual environments, Current Opinion in Biotechnology 4:450-455 (1993);Bass et al., Mutant Trp repressors with new DNA-binding specificities,Science 242:240-245 (1988); Fritz et al., Oligonucleotide-directedconstruction of mutations: a gapped duplex DNA procedure withoutenzymatic reactions in vitro, Nucl Acids Res 16: 6987-6999 (1988);Kramer et al., Improved enzymatic in vitro reactions in the gappedduplex DNA approach to oligonucleotide-directed construction ofmutations, Nucl Acids Res 16: 7207 (1988); Sakamar and Khorana, Totalsynthesis and expression of a gene for the a-subunit of bovine rod outersegment guanine nucleotide-binding protein (transducin), Nucl Acids Res14: 6361-6372 (1988); Sayers et al., Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis, Nucl AcidsRes 16:791-802 (1988); Sayers et al., Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide, (1988) Nucl Acids Res16: 803-814; Carter, Improved oligonucleotide-directed mutagenesis usingM13 vectors, Methods in Enzymol 154: 382-403 (1987); Kramer & FritzOligonucleotide-directed construction of mutations via gapped duplexDNA, Methods in Enzymol 154:350-367 (1987); Kunkel, The efficiency ofoligonucleotide directed mutagenesis, in Nucleic Acids & MolecularBiology (Eckstein, F. and Lilley, D. M. J. eds., Springer Verlag,Berlin)) (1987); Kunkel et al., Rapid and efficient site-specificmutagenesis without phenotypic selection, Methods in Enzymol 154,367-382 (1987); Zoller & Smith, Oligonucleotide-directed mutagenesis: asimple method using two oligonucleotide primers and a single-strandedDNA template, Methods in Enzymol 154:329-350 (1987); Carter,Site-directed mutagenesis, Biochem J 237:1-7 (1986); Eghtedarzadeh &Henikoff, Use of oligonucleotides to generate large deletions, NuclAcids Res 14: 5115 (1986); Mandecki, Oligonucleotide-directeddouble-strand break repair in plasmids of Escherichia coli: a method forsite-specific mutagenesis, Proc Natl Acad Sci USA, 83:7177-7181 (1986);Nakamaye & Eckstein, Inhibition of restriction endonuclease Nci Icleavage by phosphorothioate groups and its application tooligonucleotide-directed mutagenesis, Nucl Acids Res 14: 9679-9698(1986); Wells et al., Importance of hydrogen-bond formation instabilizing the transition state of subtilisin, Phil Trans R Soc Lond A317: 415-423 (1986); Botstein & Shortle, Strategies and applications ofin vitro mutagenesis, Science 229:1193-1201 (1985); Carter et al.,Improved oligonucleotide site-directed mutagenesis using M13 vectors,Nucl Acids Res 13: 4431-4443 (1985); Grundst{umlaut over (r)}om et al.,Oligonucleotide-directed mutagenesis by microscale ‘shot-gun’ genesynthesis, Nucl Acids Res 13: 3305-3316 (1985); Kunkel, Rapid andefficient site-specific mutagenesis without phenotypic selection, ProcNatl Acad Sci USA 82:488-492 (1985); Smith, In vitro mutagenesis, AnnRev Genet. 19:423-462 (1985); Taylor et al., The use ofphosphorothioate-modified DNA in restriction enzyme reactions to preparenicked DNA, Nucl Acids Res 13: 8749-8764 (1985); Taylor et al., Therapid generation of oligonucleotide-directed mutations at high frequencyusing phosphorothioate-modified DNA, Nucl Acids Res 13: 8765-8787(1985); Wells et al., Cassette mutagenesis: an efficient method forgeneration of multiple mutations at defined sites, Gene 34:315-323(1985); Kramer et al., The gapped duplex DNA approach tooligonucleotide-directed mutation construction, Nucl Acids Res 12:9441-9456 (1984); Kramer et al., Point Mismatch Repair, Cell 38:879-887(1984); Nambiar et al., Total synthesis and cloning of a gene coding forthe ribonuclease S protein, Science 223: 1299-1301 (1984); Zoller &Smith, Oligonucleotide-directed mutagenesis of DNA fragments cloned intoM13 vectors, Methods in Enzymol 100:468-500 (1983); and Zoller & Smith,Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment, Nucl Acids Res 10:6487-6500 (1982). Additional detailson many of the above methods can be found in Methods in Enzymol Volume154, which also describes useful controls for trouble-shooting problemswith various mutagenesis, gene isolation, expression, and other methods.

Oligonucleotides, e.g., for use in mutagenesis of the present invention,e.g., mutating libraries of the HA and/or NA molecules of the invention,or altering such, are typically synthesized chemically according to thesolid phase phosphoramidite triester method described by Beaucage andCaruthers, Tetrahedron Letts 22(20):1859-1862, (1981) e.g., using anautomated synthesizer, as described in Needham-VanDevanter et al.,Nucleic Acids Res, 12:6159-6168 (1984).

In addition, essentially any nucleic acid can be custom or standardordered from any of a variety of commercial sources, such as The MidlandCertified Reagent Company, The Great American Gene Company (see theWorld Wide Web at the URL “genco.com”), ExpressGen Inc. (see the WorldWide Web at the URL “expressgen.com”), Operon Technologies Inc.(Alameda, Calif.) and many others. Similarly, peptides and antibodiescan be custom ordered from any of a variety of sources, such asPeptidoGenic, HTI Bio-products, Inc. (see the World Wide Web at the URL“htibio.com”), BMA Biomedicals Ltd. (U.K.), Bio. Synthesis, Inc., andmany others.

The present invention also relates to host cells and organismscomprising a HA and/or NA molecule or other polypeptide and/or nucleicacid of the invention or such HA and/or NA or other sequences withinvarious vectors such as 6:2 reassortant influenza viruses, plasmids inplasmid rescue systems, etc. Host cells are genetically engineered(e.g., transformed, transduced or transfected) with the vectors of thisinvention, which can be, for example, a cloning vector or an expressionvector. The vector can be, for example, in the form of a plasmid, abacterium, a virus, a naked polynucleotide, or a conjugatedpolynucleotide. The vectors are introduced into cells and/ormicroorganisms by standard methods including electroporation (see, Fromet al., Proc Natl Acad Sci USA 82, 5824 (1985), infection by viralvectors, high velocity ballistic penetration by small particles with thenucleic acid either within the matrix of small beads or particles, or onthe surface (Klein et al., Nature 327, 70-73 (1987)). Berger, Sambrook,and Ausubel provide a variety of appropriate transformation methods.See, above.

Several well-known methods of introducing target nucleic acids intobacterial cells are available, any of which can be used in the presentinvention. These include: fusion of the recipient cells with bacterialprotoplasts containing the DNA, electroporation, projectile bombardment,and infection with viral vectors, etc. Bacterial cells can be used toamplify the number of plasmids containing DNA constructs of thisinvention. The bacteria are grown to log phase and the plasmids withinthe bacteria can be isolated by a variety of methods known in the art(see, for instance, Sambrook). In addition, a plethora of kits arecommercially available for the purification of plasmids from bacteria,(see, e.g., EasyPrep™, FlexiPrep™, both from Pharmacia Biotech;StrataClean™, from Stratagene; and, QIAprep™ from Qiagen). The isolatedand purified plasmids are then further manipulated to produce otherplasmids, used to transfect cells or incorporated into related vectorsto infect organisms. Typical vectors contain transcription andtranslation terminators, transcription and translation initiationsequences, and promoters useful for regulation of the expression of theparticular target nucleic acid. The vectors optionally comprise genericexpression cassettes containing at least one independent terminatorsequence, sequences permitting replication of the cassette ineukaryotes, or prokaryotes, or both, (e.g., shuttle vectors) andselection markers for both prokaryotic and eukaryotic systems. Vectorsare suitable for replication and integration in prokaryotes, eukaryotes,or preferably both. See, Giliman & Smith, Gene 8:81 (1979); Roberts, etal., Nature, 328:731 (1987); Schneider, B., et al., Protein Expr Purif6435:10 (1995); Ausubel, Sambrook, Berger (all supra). A catalogue ofBacteria and Bacteriophages useful for cloning is provided, e.g., by theATCC, e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1992)Gherna et al. (eds.) published by the ATCC. Additional basic proceduresfor sequencing, cloning and other aspects of molecular biology andunderlying theoretical considerations are also found in Watson et al.(1992) Recombinant DNA Second Edition Scientific American Books, NY.See, above.

Polypeptide Production and Recovery

In some embodiments, following transduction of a suitable host cell lineor strain and growth of the host cells to an appropriate cell density, aselected promoter is induced by appropriate means (e.g., temperatureshift or chemical induction) and cells are cultured for an additionalperiod. In some embodiments, a secreted polypeptide product, e.g., a HAand/or NA polypeptide as in a secreted fusion protein form, etc., isthen recovered from the culture medium. In other embodiments, a virusparticle containing one or more HA and/or NA polypeptide of theinvention is produced from the cell. Alternatively, cells can beharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Eukaryotic or microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, orother methods, which are well know to those skilled in the art.Additionally, cells expressing a HA and/or a NA polypeptide product ofthe invention can be utilized without separating the polypeptide fromthe cell. In such situations, the polypeptide of the invention isoptionally expressed on the cell surface and is examined thus (e.g., byhaving HA and/or NA molecules, or fragments thereof, e.g., comprisingfusion proteins or the like) on the cell surface bind antibodies, etc.Such cells are also features of the invention.

Expressed polypeptides can be recovered and purified from recombinantcell cultures by any of a number of methods well known in the art,including ammonium sulfate or ethanol precipitation, acid extraction,anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography (e.g., using any of the tagging systems known to thoseskilled in the art), hydroxylapatite chromatography, and lectinchromatography. Protein refolding steps can be used, as desired, incompleting configuration of the mature protein. Also, high performanceliquid chromatography (HPLC) can be employed in the final purificationsteps. In addition to the references noted herein, a variety ofpurification methods are well known in the art, including, e.g., thoseset forth in Sandana (1997) Bioseparation of Proteins, Academic Press,Inc.; and Bollag et al. (1996) Protein Methods, 2^(nd) EditionWiley-Liss, NY; Walker (1996) The Protein Protocols Handbook HumanaPress, NJ, Harris and Angal (1990) Protein Purification Applications: APractical Approach IRL Press at Oxford, Oxford, England; Harris andAngal Protein Purification Methods: A Practical Approach IRL Press atOxford, Oxford, England; Scopes (1993) Protein Purification: Principlesand Practice 3^(rd) Edition Springer Verlag, NY; Janson and Ryden (1998)Protein Purification: Principles, High Resolution Methods andApplications, Second Edition Wiley-VCH, NY; and Walker (1998) ProteinProtocols on CD-ROM Humana Press, NJ.

When the expressed polypeptides of the invention are produced inviruses, the viruses are typically recovered from the culture medium, inwhich infected (transfected) cells have been grown. Typically, crudemedium is clarified prior to concentration of influenza viruses. Commonmethods include ultrafiltration, adsorption on barium sulfate andelution, and centrifugation. For example, crude medium from infectedcultures can first be clarified by centrifugation at, e.g., 1000-2000×gfor a time sufficient to remove cell debris and other large particulatematter, e.g., between 10 and 30 minutes. Optionally, the clarifiedmedium supernatant is then centrifuged to pellet the influenza viruses,e.g., at 15,000×g, for approximately 3-5 hours. Following resuspensionof the virus pellet in an appropriate buffer, such as STE (0.01 MTris-HCl; 0.15 M NaCl; 0.0001 M EDTA) or phosphate buffered saline (PBS)at pH 7.4, the virus is concentrated by density gradient centrifugationon sucrose (60%-12%) or potassium tartrate (50%-10%). Either continuousor step gradients, e.g., a sucrose gradient between 12% and 60% in four12% steps, are suitable. The gradients are centrifuged at a speed, andfor a time, sufficient for the viruses to concentrate into a visibleband for recovery. Alternatively, and for most large-scale commercialapplications, virus is elutriated from density gradients using azonal-centrifuge rotor operating in continuous mode. Additional detailssufficient to guide one of skill through the preparation of influenzaviruses from tissue culture are provided, e.g., in Furminger. VaccineProduction, in Nicholson et al. (eds.) Textbook of Influenza pp.324-332; Merten et al. (1996) Production of influenza virus in cellcultures for vaccine preparation, in Cohen & Shafferman (eds.) NovelStrategies in Design and Production of Vaccines pp. 141-151, and U.S.Pat. No. 5,690,937. If desired, the recovered viruses can be stored at−80° C. in the presence of sucrose-phosphate-glutamate (SPG) as astabilizer

Alternatively, cell-free transcription/translation systems can beemployed to produce polypeptides comprising an amino acid sequence orsubsequence of, e.g., SEQ ID NO:49 through SEQ ID NO:96, or encoded bythe polynucleotide sequences of the invention. A number of suitable invitro transcription and translation systems are commercially available.A general guide to in vitro transcription and translation protocols isfound in Tymms (1995) In vitro Transcription and Translation Protocols:Methods in Molecular Biology Volume 37, Garland Publishing, NY.

In addition, the polypeptides, or subsequences thereof, e.g.,subsequences comprising antigenic peptides, can be produced manually orby using an automated system, by direct peptide synthesis usingsolid-phase techniques (see, Stewart et al. (1969) Solid-Phase PeptideSynthesis, WH Freeman Co, San Francisco; Merrifield J (1963) J Am ChemSoc 85:2149-2154). Exemplary automated systems include the AppliedBiosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City, Calif.).If desired, subsequences can be chemically synthesized separately, andcombined using chemical methods to provide full-length polypeptides.

Modified Amino Acids

Expressed polypeptides of the invention can contain one or more modifiedamino acids. The presence of modified amino acids can be advantageousin, for example, (a) increasing polypeptide serum half-life, (b)reducing/increasing polypeptide antigenicity, (c) increasing polypeptidestorage stability, etc. Amino acid(s) are modified, for example,co-translationally or post-translationally during recombinant production(e.g., N-linked glycosylation at N-X-S/T motifs during expression inmammalian cells) or modified by synthetic means (e.g., via PEGylation).

Non-limiting examples of a modified amino acid include a glycosylatedamino acid, a sulfated amino acid, a prenlyated (e.g., farnesylated,geranylgeranylated) amino acid, an acetylated amino acid, an acylatedamino acid, a PEG-ylated amino acid, a biotinylated amino acid, acarboxylated amino acid, a phosphorylated amino acid, and the like, aswell as amino acids modified by conjugation to, e.g., lipid moieties orother organic derivatizing agents. References adequate to guide one ofskill in the modification of amino acids are replete throughout theliterature. Example protocols are found in Walker (1998) ProteinProtocols on CD-ROM Human Press, Towata, N.J.

Fusion Proteins

The present invention also provides fusion proteins comprising fusionsof the sequences of the invention (e.g., encoding HA and/or NApolypeptides) or fragments thereof with, e.g., immunoglobulins (orportions thereof), sequences encoding, e.g., GFP (green fluorescentprotein), or other similar markers, etc. Nucleotide sequences encodingsuch fusion proteins are another aspect of the invention. Fusionproteins of the invention are optionally used for, e.g., similarapplications (including, e.g., therapeutic, prophylactic, diagnostic,experimental, etc. applications as described herein) as the non-fusionproteins of the invention. In addition to fusion with immunoglobulinsequences and marker sequences, the proteins of the invention are alsooptionally fused with, e.g., sequences which allow sorting of the fusionproteins and/or targeting of the fusion proteins to specific cell types,regions, etc.

Antibodies

The polypeptides of the invention can be used to produce antibodiesspecific for the polypeptides given herein and/or polypeptides encodedby the polynucleotides of the invention, e.g., those shown herein, andconservative variants thereof. Antibodies specific for the abovementioned polypeptides are useful, e.g., for diagnostic and therapeuticpurposes, e.g., related to the activity, distribution, and expression oftarget polypeptides. For example, such antibodies can optionally beutilized to define other viruses within the same strain(s) as the HA/NAsequences herein.

Antibodies specific for the polypeptides of the invention can begenerated by methods well known in the art. Such antibodies can include,but are not limited to, polyclonal, monoclonal, chimeric, humanized,single chain, Fab fragments and fragments produced by an Fab expressionlibrary.

Polypeptides do not require biological activity for antibody production(e.g., full length functional hemagglutinin or neuraminidase is notrequired). However, the polypeptide or oligopeptide must be antigenic.Peptides used to induce specific antibodies typically have an amino acidsequence of at least about 4 amino acids, and often at least 5 or 10amino acids. Short stretches of a polypeptide can be fused with anotherprotein, such as keyhole limpet hemocyanin, and antibody producedagainst the chimeric molecule.

Numerous methods for producing polyclonal and monoclonal antibodies areknown to those of skill in the art, and can be adapted to produceantibodies specific for the polypeptides of the invention, and/orencoded by the polynucleotide sequences of the invention, etc. See,e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY;Paul (ed.) (1998) Fundamental Immunology, Fourth Edition,Lippincott-Raven, Lippincott Williams & Wilkins; Harlow and Lane (1989)Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY; Stites etal. (eds.) Basic and Clinical Immunology (4th ed.) Lange MedicalPublications, Los Altos, Calif., and references cited therein; Goding(1986) Monoclonal Antibodies: Principles and Practice (2d ed.) AcademicPress, New York, N.Y.; and Kohler and Milstein (1975) Nature 256:495-497. Other suitable techniques for antibody preparation includeselection of libraries of recombinant antibodies in phage or similarvectors. See, Huse et al. (1989) Science 246: 1275-1281; and Ward, etal. (1989) Nature 341: 544-546. Specific monoclonal and polyclonalantibodies and antisera will usually bind with a K_(D) of, e.g., atleast about 0.1 μM, at least about 0.01 μM or better, and, typically andat least about 0.001 μM or better.

For certain therapeutic applications, humanized antibodies aredesirable. Detailed methods for preparation of chimeric (humanized)antibodies can be found in U.S. Pat. No. 5,482,856. Additional detailson humanization and other antibody production and engineering techniquescan be found in Borrebaeck (ed.) (1995) Antibody Engineering, 2^(nd)Edition Freeman and Company, NY (Borrebaeck); McCafferty et al. (1996)Antibody Engineering, A Practical Approach IRL at Oxford Press, Oxford,England (McCafferty), and Paul (1995) Antibody Engineering ProtocolsHumana Press, Towata, N.J. (Paul). Additional details regarding specificprocedures can be found, e.g., in Ostberg et al. (1983), Hybridoma 2:361-367, Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al., U.S.Pat. No. 4,634,666.

Defining Polypeptides by Immunoreactivity

Because the polypeptides of the invention provide a variety of newpolypeptide sequences (e.g., comprising HA and NA molecules), thepolypeptides also provide new structural features which can berecognized, e.g., in immunological assays. The generation of antiserawhich specifically bind the polypeptides of the invention, as well asthe polypeptides which are bound by such antisera, are features of theinvention.

For example, the invention includes polypeptides (e.g., HA and NAmolecules) that specifically bind to or that are specificallyimmunoreactive with an antibody or antisera generated against animmunogen comprising an amino acid sequence selected from one or more ofthe sequences given herein such as in SEQ ID NOs: 49-96, etc. Toeliminate cross-reactivity with other homologues, the antibody orantisera is subtracted with the HA and/or NA molecules found in publicdatabases at the time of filing, e.g., the “control” polypeptide(s).Where the other control sequences correspond to a nucleic acid, apolypeptide encoded by the nucleic acid is generated and used forantibody/antisera subtraction purposes.

In one typical format, the immunoassay uses a polyclonal antiserum whichwas raised against one or more polypeptide comprising one or more of thesequences corresponding to the sequences herein, etc. or a substantialsubsequence thereof (i.e., at least about 30% of the full lengthsequence provided). The set of potential polypeptide immunogens derivedfrom the present sequences are collectively referred to below as “theimmunogenic polypeptides.” The resulting antisera is optionally selectedto have low cross-reactivity against the control hemagglutinin and/orneuraminidase homologues and any such cross-reactivity is removed, e.g.,by immunoabsorbtion, with one or more of the control hemagglutinin andneuraminidase homologues, prior to use of the polyclonal antiserum inthe immunoassay.

In order to produce antisera for use in an immunoassay, one or more ofthe immunogenic polypeptides is produced and purified as describedherein. For example, recombinant protein can be produced in arecombinant cell. An inbred strain of mice (used in this assay becauseresults are more reproducible due to the virtual genetic identity of themice) is immunized with the immunogenic protein(s) in combination with astandard adjuvant, such as Freund's adjuvant, and a standard mouseimmunization protocol (see, e.g., Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York, for astandard description of antibody generation, immunoassay formats andconditions that can be used to determine specific immunoreactivity).Additional references and discussion of antibodies is also found hereinand can be applied here to defining polypeptides by immunoreactivity.Alternatively, one or more synthetic or recombinant polypeptide derivedfrom the sequences disclosed herein is conjugated to a carrier proteinand used as an immunogen.

Polyclonal sera are collected and titered against the immunogenicpolypeptide in an immunoassay, for example, a solid phase immunoassaywith one or more of the immunogenic proteins immobilized on a solidsupport. Polyclonal antisera with a titer of 10⁶ or greater areselected, pooled and subtracted with the control hemagglutinin and/orneuraminidase polypeptide(s) to produce subtracted pooled titeredpolyclonal antisera.

The subtracted pooled titered polyclonal antisera are tested for crossreactivity against the control homologue(s) in a comparativeimmunoassay. In this comparative assay, discriminatory bindingconditions are determined for the subtracted titered polyclonal antiserawhich result in at least about a 5-10 fold higher signal to noise ratiofor binding of the titered polyclonal antisera to the immunogenicpolypeptides as compared to binding to the control homologues. That is,the stringency of the binding reaction is adjusted by the addition ofnon-specific competitors such as albumin or non-fat dry milk, and/or byadjusting salt conditions, temperature, and/or the like. These bindingconditions are used in subsequent assays for determining whether a testpolypeptide (a polypeptide being compared to the immunogenicpolypeptides and/or the control polypeptides) is specifically bound bythe pooled subtracted polyclonal antisera. In particular, testpolypeptides which show at least a 2-5× higher signal to noise ratiothan the control receptor homologues under discriminatory bindingconditions, and at least about a ½ signal to noise ratio as compared tothe immunogenic polypeptide(s), shares substantial structural similaritywith the immunogenic polypeptide as compared to the known receptor,etc., and is, therefore a polypeptide of the invention.

In another example, immunoassays in the competitive binding format areused for detection of a test polypeptide. For example, as noted,cross-reacting antibodies are removed from the pooled antisera mixtureby immunoabsorbtion with the control polypeptides. The immunogenicpolypeptide(s) are then immobilized to a solid support which is exposedto the subtracted pooled antisera. Test proteins are added to the assayto compete for binding to the pooled subtracted antisera. The ability ofthe test protein(s) to compete for binding to the pooled subtractedantisera as compared to the immobilized protein(s) is compared to theability of the immunogenic polypeptide(s) added to the assay to competefor binding (the immunogenic polypeptides compete effectively with theimmobilized immunogenic polypeptides for binding to the pooledantisera). The percent cross-reactivity for the test proteins iscalculated, using standard calculations.

In a parallel assay, the ability of the control protein(s) to competefor binding to the pooled subtracted antisera is optionally determinedas compared to the ability of the immunogenic polypeptide(s) to competefor binding to the antisera. Again, the percent cross-reactivity for thecontrol polypeptide(s) is calculated, using standard calculations. Wherethe percent cross-reactivity is at least 5-10× as high for the testpolypeptides as compared to the control polypeptide(s) and or where thebinding of the test polypeptides is approximately in the range of thebinding of the immunogenic polypeptides, the test polypeptides are saidto specifically bind the pooled subtracted antisera.

In general, the immunoabsorbed and pooled antisera can be used in acompetitive binding immunoassay as described herein to compare any testpolypeptide to the immunogenic and/or control polypeptide(s). In orderto make this comparison, the immunogenic, test and control polypeptidesare each assayed at a wide range of concentrations and the amount ofeach polypeptide required to inhibit 50% of the binding of thesubtracted antisera to, e.g., an immobilized control, test orimmunogenic protein is determined using standard techniques. If theamount of the test polypeptide required for binding in the competitiveassay is less than twice the amount of the immunogenic polypeptide thatis required, then the test polypeptide is said to specifically bind toan antibody generated to the immunogenic protein, provided the amount isat least about 5-10× as high as for the control polypeptide.

As an additional determination of specificity, the pooled antisera isoptionally fully immunosorbed with the immunogenic polypeptide(s)(rather than the control polypeptide(s)) until little or no binding ofthe resulting immunogenic polypeptide subtracted pooled antisera to theimmunogenic polypeptide(s) used in the immunosorbtion is detectable.This fully immunosorbed antisera is then tested for reactivity with thetest polypeptide. If little or no reactivity is observed (i.e., no morethan 2× the signal to noise ratio observed for binding of the fullyimmunosorbed antisera to the immunogenic polypeptide), then the testpolypeptide is specifically bound by the antisera elicited by theimmunogenic protein.

Nucleic Acid and Polypeptide Sequence Variants

As described herein, the invention provides for nucleic acidpolynucleotide sequences and polypeptide amino acid sequences, e.g.,hemagglutinin and neuraminidase sequences, and, e.g., compositions andmethods comprising said sequences. Examples of said sequences aredisclosed herein. However, one of skill in the art will appreciate thatthe invention is not necessarily limited to those sequences disclosedherein and that the present invention also provides many related andunrelated sequences with the functions described herein, e.g., encodinga HA and/or a NA molecule.

One of skill will also appreciate that many variants of the disclosedsequences are included in the invention. For example, conservativevariations of the disclosed sequences that yield a functionallyidentical sequence are included in the invention. Variants of thenucleic acid polynucleotide sequences, wherein the variants hybridize toat least one disclosed sequence, are considered to be included in theinvention. Unique subsequences of the sequences disclosed herein, asdetermined by, e.g., standard sequence comparison techniques, are alsoincluded in the invention.

Silent Variations

Due to the degeneracy of the genetic code, any of a variety of nucleicacid sequences encoding polypeptides and/or viruses of the invention areoptionally produced, some which can bear lower levels of sequenceidentity to the HA and NA nucleic acid and polypeptide sequences herein.The following provides a typical codon table specifying the geneticcode, found in many biology and biochemistry texts.

TABLE 1 Amino acids Codon Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg RAGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU ThreonineThr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

The codon table shows that many amino acids are encoded by more than onecodon. For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU allencode the amino acid arginine. Thus, at every position in the nucleicacids of the invention where an arginine is specified by a codon, thecodon can be altered to any of the corresponding codons described abovewithout altering the encoded polypeptide. It is understood that U in anRNA sequence corresponds to T in a DNA sequence.

Such “silent variations” are one species of “conservatively modifiedvariations,” discussed below. One of skill will recognize that eachcodon in a nucleic acid (except ATG, which is ordinarily the only codonfor methionine, and TTG, which is ordinarily the only codon fortryptophan) can be modified by standard techniques to encode afunctionally identical polypeptide. Accordingly, each silent variationof a nucleic acid which encodes a polypeptide is implicit in anydescribed sequence. The invention, therefore, explicitly provides eachand every possible variation of a nucleic acid sequence encoding apolypeptide of the invention that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code (e.g., as setforth in Table 1, or as is commonly available in the art) as applied tothe nucleic acid sequence encoding a hemagglutinin or a neuraminidasepolypeptide of the invention. All such variations of every nucleic acidherein are specifically provided and described by consideration of thesequence in combination with the genetic code. One of skill is fullyable to make these silent substitutions using the methods herein.

Conservative Variations

Owing to the degeneracy of the genetic code, “silent substitutions”(i.e., substitutions in a nucleic acid sequence which do not result inan alteration in an encoded polypeptide) are an implied feature of everynucleic acid sequence of the invention which encodes an amino acid.Similarly, “conservative amino acid substitutions,” in one or a fewamino acids in an amino acid sequence are substituted with differentamino acids with highly similar properties, are also readily identifiedas being highly similar to a disclosed construct such as those herein.Such conservative variations of each disclosed sequence are a feature ofthe present invention.

“Conservative variation” of a particular nucleic acid sequence refers tothose nucleic acids which encode identical or essentially identicalamino acid sequences, or, where the nucleic acid does not encode anamino acid sequence, to essentially identical sequences, see, Table 2below. One of skill will recognize that individual substitutions,deletions or additions which alter, add or delete a single amino acid ora small percentage of amino acids (typically less than 5%, moretypically less than 4%, 3%, 2% or 1%) in an encoded sequence are“conservatively modified variations” where the alterations result in thedeletion of an amino acid, addition of an amino acid, or substitution ofan amino acid with a chemically similar amino acid. Thus, “conservativevariations” of a listed polypeptide sequence of the present inventioninclude substitutions of a small percentage, typically less than 5%,more typically less than 4%, 3%, 2% or 1%, of the amino acids of thepolypeptide sequence, with a conservatively selected amino acid of thesame conservative substitution group. Finally, the addition of sequenceswhich do not alter the encoded activity of a nucleic acid molecule, suchas the addition of a non-functional sequence, is a conservativevariation of the basic nucleic acid.

TABLE 2 Conservative Substitution Groups 1 Alanine (A) Serine (S)Threonine (T) 2 Aspartic acid (D) Glutamic acid (E) 3 Asparagine (N)Glutamine (Q) 4 Arginine (R) Lysine (K) 5 Isoleucine (I) Leucine (L)Methionine (M) Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan(W)

Unique Polypeptide and Polynucleotide Subsequences

In one aspect, the invention provides a nucleic acid which comprises aunique subsequence in a nucleic acid selected from the sequence of HAand NA molecules disclosed herein (e.g., SEQ ID NO:1-48). The uniquesubsequence is unique as compared to a nucleic acids corresponding tonucleic acids such as, e.g., those found in GenBank or other similarpublic databases at the time of filing (e.g., other known orcharacterized hemagglutinin and/or neuraminidase nucleic acidmolecules). Alignment can be performed using, e.g., BLAST set to defaultparameters. Any unique subsequence is useful, e.g., as a probe toidentify the nucleic acids of the invention. See, above.

Similarly, the invention includes a polypeptide (e.g., from SEQ ID NO:49through 96) which comprises a unique subsequence in a polypeptideselected from the sequence of HA and NA molecules disclosed herein.Here, the unique subsequence is unique as compared to a polypeptidecorresponding to, e.g., the amino acid corresponding to polynucleotidesequences found in, e.g., GenBank or other similar public databases atthe time of filing.

The invention also provides for target nucleic acids which hybridizeunder stringent conditions to a unique coding oligonucleotide whichencodes a unique subsequence in a polypeptide selected from thesequences of HA and NA molecules of the invention wherein the uniquesubsequence is unique as compared to a polypeptide corresponding to anyof the control polypeptides (sequences of, e.g., the nucleic acidscorresponding to those found in, e.g., GenBank or other similar publicdatabases at the time of filing). Unique sequences are determined asnoted above. The polynucleotides of the invention also comprise RNA(both positive sense and negative sense) versions of the sequences ofthe sequence listing. See above.

Sequence Comparison, Identity, and Homology

The terms “identical” or percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms described below (or otheralgorithms available to persons of skill) or by visual inspection.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides (e.g., DNAs and/or RNAs encoding a HA or NAmolecule, or the amino acid sequence of a HA or NA molecule) refers totwo or more sequences or subsequences that have at least about 90%,preferably 91%, most preferably 92%, 93%, 94%, 95%, 96%, 97%, 98%,98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,99.9% or more nucleotide or amino acid residue identity, when comparedand aligned for maximum correspondence, as measured using a sequencecomparison algorithm or by visual inspection. Such “substantiallyidentical” sequences are typically considered to be “homologous,”without reference to actual ancestry. Preferably, “substantial identity”exists over a region of the amino acid sequences that is at least about200 residues in length, more preferably over a region of at least about250 residues, and most preferably the sequences are substantiallyidentical over at least about 300 residues, 350 residues, 400 residues,425 residues, 450 residues, 475 residues, 480 residues, 490 residues,495 residues, 499 residues, 500 residues, 502 residues, 559 residues,565 residues, or 566 residues, or over the full length of the twosequences to be compared when the amino acids are hemagglutinin orhemagglutinin fragments or which is substantially identical over atleast about 350 amino acids; over at least about 400 amino acids; overat least about over at least about 436 amino acids, over at least about450 amino acids; over at least about 451 amino acids; over at leastabout 465 amino acids; over at least about 466 amino acids; over atleast about 469 amino acids; over at least about 470 amino acids; orover at least about 566 amino acids contiguous when the amino acid isneuraminidase or a neuraminidase fragment.

For sequence comparison and homology determination, typically onesequence acts as a reference sequence to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv Appl Math 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J MolBiol 48:443 (1970), by the search for similarity method of Pearson &Lipman, Proc Natl Acad Sci USA 85:2444 (1988), by computerizedimplementations of algorithms such as GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis., or by visual inspection (see generally,Ausubel et al., supra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J Mol Biol 215:403-410 (1990). Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (www.ncbi.nlm.nih.gov/). Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (see, Altschul etal., supra). These initial neighborhood word hits act as seeds forinitiating searches to find longer HSPs containing them. The word hitsare then extended in both directions along each sequence for as far asthe cumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see, Henikoff & Henikoff (1989) Proc NatlAcad Sci USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc Natl Acad Sci USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

Another example of a useful sequence alignment algorithm is PILEUP.PILEUP creates a multiple sequence alignment from a group of relatedsequences using progressive, pairwise alignments. It can also plot atree showing the clustering relationships used to create the alignment.PILEUP uses a simplification of the progressive alignment method of Feng& Doolittle (1987) J. Mol. Evol. 35:351-360. The method used is similarto the method described by Higgins & Sharp (1989) CABIOS5:151-153. Theprogram can align, e.g., up to 300 sequences of a maximum length of5,000 letters. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster can then be aligned to the next mostrelated sequence or cluster of aligned sequences. Two clusters ofsequences can be aligned by a simple extension of the pairwise alignmentof two individual sequences. The final alignment is achieved by a seriesof progressive, pairwise alignments. The program can also be used toplot a dendogram or tree representation of clustering relationships. Theprogram is run by designating specific sequences and their amino acid ornucleotide coordinates for regions of sequence comparison.

An additional example of an algorithm that is suitable for multiplenucleic acid, or amino acid, sequence alignments is the CLUSTALW program(Thompson, J. D. et al. (1994) Nucl. Acids. Res. 22: 4673-4680).CLUSTALW performs multiple pairwise comparisons between groups ofsequences and assembles them into a multiple alignment based onhomology. Gap open and Gap extension penalties can be, e.g., 10 and 0.05respectively. For amino acid alignments, the BLOSUM algorithm can beused as a protein weight matrix. See, e.g., Henikoff and Henikoff (1992)Proc. Natl. Acad. Sci. USA 89: 10915-10919.

Digital Systems

The present invention provides digital systems, e.g., computers,computer readable media and integrated systems comprising characterstrings corresponding to the sequence information herein for the nucleicacids and isolated or recombinant polypeptides herein, including, e.g.,the sequences shown herein, and the various silent substitutions andconservative substitutions thereof. Integrated systems can furtherinclude, e.g., gene synthesis equipment for making genes correspondingto the character strings.

Various methods known in the art can be used to detect homology orsimilarity between different character strings (see above), or can beused to perform other desirable functions such as to control outputfiles, provide the basis for making presentations of informationincluding the sequences and the like. Examples include BLAST, discussedsupra. Computer systems of the invention can include such programs,e.g., in conjunction with one or more data file or data base comprisinga sequence as noted herein.

Thus, different types of homology and similarity of various stringencyand length between various HA or NA sequences or fragments, etc. can bedetected and recognized in the integrated systems herein. For example,many homology determination methods have been designed for comparativeanalysis of sequences of biopolymers, for spell-checking in wordprocessing, and for data retrieval from various databases. With anunderstanding of double-helix pair-wise complement interactions amongfour principal nucleobases in natural polynucleotides, models thatsimulate annealing of complementary homologous polynucleotide stringscan also be used as a foundation of sequence alignment or otheroperations typically performed on the character strings corresponding tothe sequences herein (e.g., word-processing manipulations, constructionof figures comprising sequence or subsequence character strings, outputtables, etc.).

Thus, standard desktop applications such as word processing software(e.g., Microsoft Word™ or Corel WordPerfect™) and database software(e.g., spreadsheet software such as Microsoft Excel™, Corel QuattroPro™, or database programs such as Microsoft Access™, Paradox™,GeneWorks™, or MacVector™ or other similar programs) can be adapted tothe present invention by inputting a character string corresponding toone or more polynucleotides and polypeptides of the invention (eithernucleic acids or proteins, or both). For example, a system of theinvention can include the foregoing software having the appropriatecharacter string information, e.g., used in conjunction with a userinterface (e.g., a GUI in a standard operating system such as a Windows,Macintosh or LINUX system) to manipulate strings of characterscorresponding to the sequences herein. As noted, specialized alignmentprograms such as BLAST can also be incorporated into the systems of theinvention for alignment of nucleic acids or proteins (or correspondingcharacter strings).

Systems in the present invention typically include a digital computerwith data sets entered into the software system comprising any of thesequences herein. The computer can be, e.g., a PC (Intel x86 or Pentiumchip-compatible DOS™, OS2™ WINDOWS™ WINDOWSNT™, WINDOWS95™,WINDOWS2000™, WINDOWS98™, LINUX based machine, a MACINTOSH™, Power PC,or a UNIX based (e.g., SUN™ work station) machine) or other commerciallyavailable computer that is known to one of skill. Software for aligningor otherwise manipulating sequences is available, or can easily beconstructed by one of skill using a standard programming language suchas Visualbasic, PERL, Fortran, Basic, Java, or the like.

Any controller or computer optionally includes a monitor which is oftena cathode ray tube (“CRT”) display, a flat panel display (e.g., activematrix liquid crystal display, liquid crystal display), or others.Computer circuitry is often placed in a box which includes numerousintegrated circuit chips, such as a microprocessor, memory, interfacecircuits, and others. The box also optionally includes a hard diskdrive, a floppy disk drive, a high capacity removable drive such as awriteable CD-ROM, and other common peripheral elements. Inputtingdevices such as a keyboard or mouse optionally provide for input from auser and for user selection of sequences to be compared or otherwisemanipulated in the relevant computer system.

The computer typically includes appropriate software for receiving userinstructions, either in the form of user input into a set parameterfields, e.g., in a GUI, or in the form of preprogrammed instructions,e.g., preprogrammed for a variety of different specific operations. Thesoftware then converts these instructions to appropriate language forinstructing the operation, e.g., of appropriate mechanisms or transportcontrollers to carry out the desired operation. The software can alsoinclude output elements for controlling nucleic acid synthesis (e.g.,based upon a sequence or an alignment of sequences herein), comparisonsof samples for differential gene expression, or other operations.

Kits and Reagents

The present invention is optionally provided to a user as a kit. Forexample, a kit of the invention contains one or more nucleic acid,polypeptide, antibody, or cell line described herein (e.g., comprising,or with, a HA and/or NA molecule of the invention). The kit can containa diagnostic nucleic acid or polypeptide, e.g., antibody, probe set,e.g., as a cDNA micro-array packaged in a suitable container, or othernucleic acid such as one or more expression vector. The kit typicallyfurther comprises, one or more additional reagents, e.g., substrates,labels, primers, for labeling expression products, tubes and/or otheraccessories, reagents for collecting samples, buffers, hybridizationchambers, cover slips, etc. The kit optionally further comprises aninstruction set or user manual detailing preferred methods of using thekit components for discovery or application of diagnostic sets, etc.

When used according to the instructions, the kit can be used, e.g., forevaluating a disease state or condition, for evaluating effects of apharmaceutical agent or other treatment intervention on progression of adisease state or condition in a cell or organism, or for use as avaccine, etc.

In an additional aspect, the present invention provides system kitsembodying the methods, composition, systems and apparatus herein. Systemkits of the invention optionally comprise one or more of the following:(1) an apparatus, system, system component or apparatus component; (2)instructions for practicing methods described herein, and/or foroperating the apparatus or apparatus components herein and/or for usingthe compositions herein. In a further aspect, the present inventionprovides for the use of any apparatus, apparatus component, compositionor kit herein, for the practice of any method or assay herein, and/orfor the use of any apparatus or kit to practice any assay or methodherein.

Additionally, the kits can include one or more translation system asnoted above (e.g., a cell) with appropriate packaging material,containers for holding the components of the kit, instructionalmaterials for practicing the methods herein and/or the like. Similarly,products of the translation systems (e.g., proteins such as HA and/or NAmolecules) can be provided in kit form, e.g., with containers forholding the components of the kit, instructional materials forpracticing the methods herein and/or the like. Furthermore, the kits cancomprise various vaccines (e.g., produced through plasmid rescueprotocols) such as live attenuated vaccine (e.g., FluMist™) comprisingthe HA and/or NA sequences herein.

To facilitate use of the methods and compositions of the invention, anyof the vaccine components and/or compositions, e.g., reassorted virus inallantoic fluid, etc., and additional components, such as, buffer,cells, culture medium, useful for packaging and infection of influenzaviruses for experimental or therapeutic vaccine purposes, can bepackaged in the form of a kit. Typically, the kit contains, in additionto the above components, additional materials which can include, e.g.,instructions for performing the methods of the invention, packagingmaterial, and a container.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovemay be used in various combinations. All publications, patents, patentapplications, or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application, orother document were individually indicated to be incorporated byreference for all purposes.

What is claimed is:
 1. A 6:2 reassortant influenza B virus, wherein saidvirus comprises 6 internal genome segments from one or more donorviruses and two surface antigen genome segments, wherein the surfaceantigen genome segments encode an HA and a NA polypeptide, and whereinthe HA polypeptide comprises the amino acid sequence of SEQ ID NO: 93.2. The 6:2 reassortant influenza B virus of claim 1, wherein said one ormore donor viruses comprise one or more of the following phenotypes:temperature-sensitive, cold-adapted, or attenuated.
 3. The 6:2reassortant influenza B virus of claim 1, wherein said one or more donorviruses are selected from the group consisting of B/Ann Arbor/1/66,B/Leningrad/14/17/55, B/14/5/1, B/USSR/60/69, B/Leningrad/179/86,B/Leningrad/14/55, and B/England/2608/76.
 4. The 6:2 reassortantinfluenza B virus of claim 1, wherein said reassortant influenza B virusis killed or inactivated.
 5. The 6:2 reassortant influenza B virus ofclaim 1, wherein said reassortant influenza B virus is a live attenuatedreassortant influenza B virus.
 6. An immunogenic vaccine compositioncomprising an immunologically effective amount of the reassortantinfluenza B virus of claim
 2. 7. An immunogenic vaccine compositioncomprising an immunologically effective amount of the reassortantinfluenza B virus of claim
 4. 8. An immunogenic vaccine compositioncomprising an immunologically effective amount of the reassortantinfluenza B virus of claim
 5. 9. A method for stimulating the immunesystem of an individual to produce a protective immune response againstinfluenza B virus, the method comprising administering to the individualan immunologically effective amount of the reassortant influenza B virusof claim 2 in a physiologically effective carrier.
 10. A method forstimulating the immune system of an individual to produce a protectiveimmune response against influenza B virus, the method comprisingadministering to the individual an immunologically effective amount ofthe reassortant influenza B virus of claim 4 in a physiologicallyeffective carrier.
 11. A method for stimulating the immune system of anindividual to produce a protective immune response against influenza Bvirus, the method comprising administering to the individual animmunologically effective amount of the reassortant influenza B virus ofclaim 5 in a physiologically effective carrier.
 12. A method ofprophylactic or therapeutic treatment of a influenza B viral infectionin a subject, the method comprising: administering to the subject thereassortant influenza B virus of claim 2 in an amount effective toproduce an immunogenic response against the influenza B viral infection.13. A method of prophylactic or therapeutic treatment of a influenza Bviral infection in a subject, the method comprising: administering tothe subject the reassortant influenza B virus of claim 4 in an amounteffective to produce an immunogenic response against the influenza Bviral infection.
 14. A method of prophylactic or therapeutic treatmentof a influenza B viral infection in a subject, the method comprising:administering to the subject the reassortant influenza B virus of claim5 in an amount effective to produce an immunogenic response against theinfluenza B viral infection.
 15. The method of claim 12, wherein thesubject is a human.
 16. The method of claim 13, wherein the subject is ahuman.
 17. An immunogenic vaccine composition comprising animmunologically effective amount of the reassortant influenza B virus ofclaim 1, wherein the reassortant influenza virus is a split virus. 18.The immunogenic vaccine composition of claim 6, wherein the reassortantinfluenza virus is a split virus.
 19. The immunogenic vaccinecomposition of claim 7, wherein the reassortant influenza virus is asplit virus.
 20. A method for producing an influenza B virus in cellculture, the method comprising: i) introducing into a population of hostcells, which population of host cells is capable of supportingreplication of influenza virus, a plurality of vectors comprisingnucleotide sequences corresponding to: (a) at least 6 internal genomesegments of one or more donor viruses, and at least one genome segmentencoding an HA surface antigen polypeptide, wherein the surface antigenpolypeptide comprises the amino acid sequence from SEQ ID NO: 93; or (b)at least 6 internal genome segments of one or more donor viruses, whichone or more donor viruses comprises one or more phenotypic attributesselected from the group consisting of: attenuated, cold adapted andtemperature sensitive; and at least one genome segment encoding an HAsurface antigen, wherein the surface antigen polypeptide comprises theamino acid sequence of SEQ ID NO: 93; ii) culturing the population ofhost cells at a temperature less than or equal to 35° C.; and, iii)recovering influenza virus.